Nucleic acid preparation methods

ABSTRACT

The present invention provides a much simplified method for the preparation of nucleic acid samples. This method provides many advantages over prior sample preparation methods in terms of greater sensitivity, reliability and ease of use. Importantly, the present invention provides a method in which nucleic acid samples may be prepared so as to conserve reagents and time. The samples so prepared are readily amplifiable and may be used for other purposes as well.

This is a continuation of application Ser. No. 07/901,545 filed on06/19/92, now abandoned, which is a continuation-in-part of issuedapplication Ser. No. 07/614,921 filed on 11/14/90 now U.S. Pat. No.5,284,940.

FIELD OF THE INVENTION

The present invention relates to methods of nucleic acid preparation,and in particular, preparation of RNA from samples for subsequentamplification.

BACKGROUND

Problems in nucleic acid preparation due to non-nucleic acid componentsof the nucleic acid source are well-known. For example, the preparationof RNA is complicated by the presence of ribonucleases that degrade RNA.T. Maniatis et al., Molecular Cloning, pp. 188-190 (Cold Spring HarborLaboratory 1982). Furthermore, the preparation of amplifiable RNA ismade difficult by the presence of ribonucleoproteins in association withRNA. See R. J. Slater, In: Techniques in Molecular Biology, (Macmillan,N.Y. 1983) (J. M. Walker and W. Gaastra, eds.) (pp. 113-120).

Three basic methods are employed for RNA preparation: 1) extraction withphenol; 2) degradation with protease; or 3) disruption and ribonucleaseinhibition with strong salts. Phenol is basically a denaturant. Whileuseful, phenol extraction is time consuming and creates a serious wastedisposal problem. Use of protease requires the addition of a detergent(e.g. SDS); detergents must be removed for the recovered RNA to beuseful in subsequent assays. Finally, the use of salts alone does notresult in the purification of RNA that is free of protein; currentprotocols require the use of salts in conjunction with phenol [P.Chomczynski and N. Sacchi, Anal. Biochem. 162:156 (1987)] or employ acentrifugation step to remove the protein (R. J. Slater, supra).

Where purification of RNA is for the purpose of producing template foramplification, it is important to consider the source (i.e. bone marrow,spinal fluid, urine, feces, etc.) and potential polymerase inhibitorsthat are constituents in such sources. One class of constituents knownto inhibit nucleic acid associated enzymes are the "hemes" which includehemin and hematin. Hemin has been reported to inhibit virion-associatedreverse transcriptase (RTase) of murine leukemia virus (MuLV) (Tsutsuiand Mueller, BBRC 149: 628-634, 1987), DNA ligase (Scher et al., Can.Res. 48: 6278-6284, 1988), cytoplasmic DNA polymerase (Byrnes et al.,Biochem. 14, 796-799, 1975), Taq polymerase (PCR Technology, H. A.Erlich (ed.) Stockton Press (1989) p.33), and other enzymatic systemsthat utilize ATP as a cofactor such as the hemin-controlled proteinkinase that affects protein synthesis (Hronis and Traugh, J. Biol. Chem.261, 6234-6238, 1986), the ATP-dependent ubiquitin-dependent proteasepathway (Hershko et al., PNAS USA 81, 1619-1623, 1984), and theATP-dependent ubiquitin-independent protease pathway (Waxman et al., J.Biol. Chem. 260, 11994-12000, 1985).

Freshly-made hemin solution inhibited MuLV RTase activity by 50% at ahemin concentration of 10 μM. Aged hemin solutions (5 days at roomtemperature) inhibited MuLV RTase by 50% at 0.1 μM concentration.Addition of 4-fold excess MuLV RTase caused an increase of enzymeactivity in the presence of hemin while addition of excess template didnot. Addition of a heme-binding protein from rabbit serum (Tsutsui andMueller, J. Biol. Chem. 257, 3925-3931, 1982) completely restored enzymeactivity. This suggests that hemin is a reversible inhibitor of MuLVRTase and that its interaction with the enzyme is noncovalent in nature.Hemin does not inhibit the activity of reverse transcriptase purifiedfrom avian myeloblastosis virus.

Experiments with DNA ligase indicate that hemin at 4 μM or less does notaffect DNA ligase activity or DNA substrate integrity. Scher et al.,Can. Res. 48, 6278-6284, 1988. Preincubations of DNA ligase with heminled to half-maximal inhibition of DNA ligase at hemin concentrations of25-100 μM (depending on the source of the DNA ligase). NAD-dependent DNAligase from E. coli was not inhibited by hemin at concentrations up to150 μM. The inhibition of T4 DNA ligase activity and DNA ligase frommouse erythroleukemia (MEL) cells was not reversible by dilution,dialysis, or sucrose gradient centrifugation of cell-free extracts.Incubation of DNA ligase from MEL cells with hemoglobin was notinhibitory.

Binding assays demonstrate that hemin prevents association and causesdissociation of the DNA-cytoplasmic DNA polymerase complex. Hemin at aconcentration of 12 μM or higher completely inhibits the formation ofDNA-enzyme complex. Byrnes et al., Biochem. 14, 796-799, 1975. Thisreport also shows that hemin inhibition of DNA synthesis is competitivewith respect to template and noncompetitive with respect to substrate.Furthermore, inhibition could be reversed by either 1) addition ofglobin to the polymerase-containing reaction mixture prior to theaddition of hemin to the reaction mixture, or 2) addition of globin tohemin followed by addition of this mixture to the polymerase-containingreaction mixture. Inhibition could not be reversed by the addition ofglobin after introduction of hemin to the polymerase-containing reactionmixture

Experiments with purified hematin and related compounds have shown thatthey are potent inhibitors of Taq polymerase. Taq polymerase is used inthe amplification procedure described by K. B. Mullis et. al., U.S. Pat.Nos. 4,683,195 and 4,683,202. This amplification procedure is a methodfor increasing the concentration of a segment of a target sequence in amixture of genomic DNA without cloning or purification. This process foramplifying the target sequence consists of introducing a large excess oftwo oligonucleotide primers to the DNA mixture containing the desiredtarget sequence, followed by a precise sequence of thermal cycling inthe presence of a DNA polymerase. The two primers are complementary totheir respective strands of the double stranded target sequence. Toeffect amplification, the mixture is denatured and the primers thenannealed to their complementary sequences within the target molecule.Following annealing, the primers are extended with a polymerase so as toform a new pair of complementary strands. The steps of denaturation,primer annealing, and polymerase extension can be repeated many times(i.e. denaturation, annealing and extension constitute one "cycle;"there can be numerous "cycles") to obtain a high concentration of anamplified segment of the desired target sequence. The length of theamplified segment of the desired target sequence is determined by therelative positions of the primers with respect to each other, andtherefore, this length is a controllable parameter. By virtue of therepeating aspect of the process, the method is referred to by theinventors as the "Polymerase Chain Reaction" (hereinafter PCR). Becausethe desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be "PCR amplified".

Hematin is inhibitory to PCR at a final concentration of 0.8 μM orhigher (PCR Technology, H. A. Erlich (ed.) Stockton Press (1989) p. 33)."Protoporphyrin" is inhibitory at 20 μM. Non-heme blood components suchas globin, Fe++ and Fe⁺⁺⁺ ions also inhibit PCR (Walsh et al.).

Where the inhibitor is a competitive inhibitor, one approach is to addmore reagent and "swamp" the inhibition. This has been attempted in thecase of PCR inhibition. Walsh et al. have shown that, while hematininhibition cannot be overcome by additional quantities of template DNA,it can be overcome by additional quantities of Taq polymerase or primer.

This swamping approach has a serious disadvantage: additional quantitiesof reagents may cause spurious results. Indeed, in the case of PCR it isknown that additional quantities of Taq polymerase or primers can resultin nonspecific amplification products. S. Paabo et al., Nucleic AcidsRes. 16:9775 (1988). These nonspecific products are believed to be dueto weak priming sites.

The conventional method for the preparation of amplifiable nucleic acidfrom whole blood involves isolation of lymphocytes by density gradientcentrifugation. This typically involves the isolation of T4 enrichedwhite blood cells by centrifugation through a Ficoll gradient. See e.g.Longley and Stewart, J. Immunol. Methods, 121, 33-38, 1989. The redblood cells and granulocytes pellet in this system. Thelymphocyte-enriched white blood cells are recovered from the gradientinterface. To remove Ficoll, which inhibits Taq polymerase, the cellsare usually washed one or more times by centrifuging and removing thesupernatant. Although this procedure yields lymphocytes free of redblood cells and most of the platelets, there are a number ofdisadvantages to this procedure, including: (1) relatively large,freshly drawn blood samples must be layered over Ficoll carefully sothat the interface is undisturbed; (2) lymphocytes must be collected(after centrifugation) by removing the opaque band of cells located atthe gradient interface; and (3) the collected lymphocytes must be washedfree of Ficoll. The careful layering of blood is a slow and somewhatartful step. The collection of the cells at the gradient interfacedemands that i) enough blood be used initially such that the cells canbe seen with the naked eye, ii) the cells be captured in a pipette (acumbersome and low-yield technique), and iii) amplification be carriedout in a different reaction vessel from that used to layer the blood.Finally, the approach utilizes a polymerase inhibitor (i.e. Ficoll) inlarge amounts that is not easily removed except by centrifugation. Thesedrawbacks have seriously hindered the application of amplificationtechniques to large-scale clinical diagnostics.

SUMMARY OF THE INVENTION

The present invention relates to methods of nucleic acid preparation,and in particular, preparation of RNA from samples for subsequentamplification. In one embodiment, the present invention contemplates amethod of purifying and recovering nucleic acid, comprising the stepsof: a) providing, in any order, i) a sample suspected of containingnucleic acid, ii) guanidinium thiocyanate, iii) beta-mercaptoethanol,iv) an alcohol, and v) a reaction vessel; b) adding to said reactionvessel, in any order, said sample, said guanidinium thiocyanate and saidbeta-mercaptoethanol, to make a reaction mixture; c) heating saidreaction mixture; and d) adding said alcohol to recover said nucleicacid from said reaction mixture.

In a preferred embodiment, said alcohol comprises isopropanol, heatingabove 60° C., and the additional step of cooling said reaction mixtureafter step (c) prior to step (d). Cooling may be performed until thereaction mixture reaches room temperature or until the reaction mixturereaches approximately 4° C.

While it is not intended that the present invention be limited by thenature or source of the nucleic acid, success is achieved withribonucleic acid, including ribonucleic acid from a pathogen (such asHCV or HIV RNA).

Similarly, while it is not intended that the present invention belimited by the type of sample, the invention can be used with particularsuccess with human samples. Such human samples may be selected from thegroup comprising plasma, serum and spinal fluid.

The invention is particularly useful where it is desired to amplify saidnucleic acid recovered in step (d). In a preferred embodiment,amplification is performed on recovered ribonucleic acid by adding athermostable DNA polymerase having endogenous reverse transcriptaseactivity to said recovered ribonucleic acid. The method is compatiblewith the use of deoxyribonucleoside triphosphate dUTP (preferrably atapproximately 400 mM).

DESCRIPTION OF THE DRAWINGS

FIG. 1 schematically shows embodiments of the method of the presentinvention for processing whole blood for amplification.

FIG. 2A is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified HIV sequences following chemicalreduction of hematin. (Direct printing results in light bands on a darkbackground). FIG. 2B is a graph showing quantitative changes in PCRsignal following chemical reduction of hematin.

FIG. 3A is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified HIV sequences followingphotochemical treatment of biliverdin. FIG. 3B shows absorption spectraof biliverdin following photochemical treatment over time.

FIG. 4A is a photograph of an autoradiograph of electrophoresed,PCR-amplified, internally-radiolabelled, HIV sequences followingphotochemical treatment of bilirubin. FIG. 4B shows absorption spectraof bilirubin following photochemical treatment over time.

FIG. 5 schematically shows enzymatic inhibitor treatment according toone embodiment of the method of the present invention for processingwhole blood for amplification.

FIG. 6 schematically shows inhibitor treatment wherein both enzymaticand photochemical processes are employed in combination according to thepresent invention.

FIG. 7 schematically shows inhibitor treatment wherein both chemical andphotochemical processes are employed in combination according to thepresent invention.

FIG. 8 is a graph showing quantitative changes in PCR signal followinginhibitor treatment involving chemical and photochemical treatment incombination according to the present invention.

FIG. 9 is a (direct print) photograph of an ethidium bromide stained gelof electrophoresed PCR-amplified HIV sequences showing the impact of thepresence of reagents used for the selective lysis step (see FIG. 1) ofthe present invention with respect to amplification inhibition.

FIG. 10 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified HIV sequences following the stepsof Mode Ib (see FIG. 1) of the method of the present invention.

FIG. 11 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified HLA Class II gene sequencesfollowing the steps of Mode Ib (see FIG. 1) of the method of the presentinvention.

FIG. 12 is a photograph of an autoradiograph of electrophoresed,PCR-amplified, HIV sequences visualized by oligonucleotide hybridizationanalysis following the steps of Mode Ia (see FIG. 1) of the method ofthe present invention

FIG. 13 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, globin gene sequences followingthe steps of Mode III (see FIG. 1) of the method of the presentinvention.

FIG. 14A is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified HIV sequences comparing thespecificity and sensitivity of amplifications following the steps ofMode Ib and Mode III (see FIG. 1) of the method of the presentinvention. FIG. 14B is a photograph of an autoradiograph ofelectrophoresed, PCR-amplified, HIV sequences visualized byoligonucleotide hybridization analysis comparing the specificity andsensitivity of amplifications following the steps of Mode Ib and ModeIII (see FIG. 1) of the method of the present invention.

FIG. 15 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, HIV sequences followingbiochemical inhibitor treatment according to one embodiment of themethod of the present invention.

FIG. 16A, B & C are (direct print) photographs of an ethidium bromidestained gel of electrophoresed PCR-amplified, HLA class II genesequences following biochemical inhibitor treatment according toembodiments of the method of the present invention.

FIG. 17 is a photograph of a Coomassie blue stained, SDS-PAGE gel offractionated compounds of the present invention.

FIG. 18 is a photograph of an Ouchterlony immunodiffusion gel showingthe formation of precipitin lines where antibody has reacted with someof the compounds of the present invention.

FIG. 19 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, HIV sequences followingbiochemical inhibitor treatment according to one embodiment of themethod of the present invention.

FIG. 20 schematically shows one embodiment of the method of the presentinvention for making deoxyribonucleic acid from ribonucleic acidaccording processing whole blood for amplification.

FIG. 21 schematically shows one embodiment of the method of the presentinvention for recovering ribonucleic acid from a sample.

FIG. 22 shows the nucleic acid sequences (SEQ ID NO:1) for the HCVamplification employed in the use of the present invention.

FIG. 23A is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, HCV sequences following recoveryof RNA and synthesis of cDNA from patient plasma according to oneembodiment of the method of the present invention.

FIG. 23B is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, HCV sequences following recoveryof RNA and synthesis of cDNA from additional patient plasma according toone embodiment of the method of the present invention.

FIG. 24 is a (direct print) photograph of an ethidium bromide stainedgel of electrophoresed PCR-amplified, HCV sequences following recoveryof RNA and synthesis of cDNA from serum according to one embodiment ofthe method of the present invention.

DESCRIPTION OF THE INVENTION

The present invention relates to methods of nucleic acid preparation,and in particular, preparation of RNA from samples for subsequentamplification. By "samples" it is meant that the substance or mixture tobe assayed may contain more than simply biological material. While thepreferred biological sample of the present invention is cell-free (e.g.plasma, urine, serum, etc.), it is not intended that the presentinvention be limited to this source alone. For example, the presentinvention is successfully employed with spinal fluid. It should bestressed that it is not intended that the present invention be limitedto preparation of nucleic acid for amplification. The present inventionhas applicability at any point that preparation of nucleic acid samplesis desired. Nonetheless, the present invention has particularapplicability when amplification is desired (whether deoxyribonucleicacid or ribonucleic acid amplification).

It should be stressed that it is not intended that the present inventionbe limited to any particular amplification technique. "Amplification" isa special case of nucleic acid replication involving templatespecificity. It is to be contrasted with non-specific templatereplication (i.e. replication that is template-dependent but notdependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e. synthesis of the properpolynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of"target" specificity. Target sequences are "targets" in the sense thatthey are sought to be sorted out from other nucleic acid. Amplificationtechniques have been designed primarily for this sorting out.

Template specificity is achieved in most amplification techniques by thechoice of enzyme. Amplification enzymes are enzymes that, underconditions they are used, will process only specific sequences ofnucleic acid in a heterogeneous mixture of nucleic acid. For example, inthe case of Qβ replicase, MDV-1 RNA is the specific template for thereplicase. D. L. Kacian et. al., Proc. Nat. Acad. Sci USA 69:3038(1972). Other nucleic acid will not be replicated by this amplificationenzyme. Similarly, in the case of T7 RNA polymerase, this amplificationenzyme has a stringent specificity for its own promoters. M. Chamberlinet al., Nature 228:227 (1970). In the case of T4 DNA ligase, the enzymewill not ligate the two oligonucleotides where there is a mismatchbetween the oligonucleotide substrate and the template at the ligationjunction. D. Y. Wu and R. B. Wallace, Genomics 4:560 (1989). Finally,Taq polymerase, by virtue of its ability to function at hightemperature, is found to display high specificity for the sequencesbound and thus defined by the primers; the high temperature results inthermodynamic conditions that favor primer hybridization with the targetsequences and not hybridization with non-target sequences. PCRTechnology, H. A. Erlich (ed.) (Stockton Press 1989).

Some amplification techniques take the approach of amplifying and thendetecting target; others detect target and then amplify probe.Regardless of the approach, nucleic acid must be free of inhibitors foramplification to occur at high efficiency.

"Amplification reagents" are defined as those reagents [primers,standard deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, and dTTP,etc.) needed for amplification except for nucleic acid and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

"Polymerase inhibitors" include all compounds (organic and inorganic)that reduce the amount of nucleic acid replication by enzymes. It is notintended to be limited by the mechanism by which inhibitors achieve thisreduction. Furthermore, it is not intended to be limited to only thoseinhibitors which display large reductions.

The present invention offers a radical change from the cumbersomenucleic acid whole blood method of density centrifugation and provides aflexible approach to whole blood processing for nucleic acidamplification (see FIG. 1). Importantly, the method of whole bloodprocessing of the present invention can be carried out with i) onlymicroliter amounts of blood (e.g. fingerstick or heelstick), ii) storedblood (including dried blood), and iii) the same, single reaction vesselused for amplification (allowing for quantitative recovery of cellularnucleic acid, in contrast, to the low-yield density centrifugationmethod). Because of these features, the method of the present inventionis also amenable to automation.

In one mode ("Mode I"), the present invention offers a selective celllysis step that involves lysing red blood cells in whole blood ordiluted whole blood under conditions which leave white blood cellsand/or their nuclei intact. Thereafter, a process step is performed thatinvolves concentration of the cells. "Concentration" is defined broadlyand may result from a wide variety of techniques, including filtration,agglutination, centrifugation, immobilization, and/or fluid evaporation.Following the concentration step, there is another lysis step. Since thered cells are gone, the lysis need not be selective and is thereforereferred to as "non-selective" lysis. However, the present inventioncontemplates the use of selective reagents in the non-selective lysisstep as well. At this point, Mode I may or may not have another stepbefore amplification. In one version ("a"), the method proceeds to aninhibitor treatment step. By "inhibitor treatment" it is meant that aprocess is carried out to treat potential, residual polymeraseinhibitors such as heme and its related compounds. The process may bechemical, biochemical, photochemical, immunological, or enzymatic, orany combination thereof (e.g. enzymatic followed by photochemical;chemical followed by enzymatic; chemical followed by photochemical). Inanother version ("b"), the method proceeds directly to amplification.

In another mode ("Mode II"), the present invention offers aconcentration step followed by a non-selective lysis step, which in turnis followed by an inhibitor treatment step prior to amplification. Instill another mode ("Mode III"), a non-selective cell lysis step is usedfollowed by an inhibitor treatment step prior to amplification. Again,each of these steps is defined broadly (see definitions above). Each ofthese modes, and the numerous embodiments for each of these modes, aredescribed in detail below.

Mode I

Selective Cell Lysis

The present invention contemplates a selective cell lysis step thatinvolves lysing red blood cells in whole blood or diluted whole bloodunder conditions which leave white blood cells and/or their nucleiintact. The present invention contemplates a wide variety of selectivelysing agents. For example, the present invention contemplates selectivelysis by the use of agents such as saponin, dilute hydrochloric acid,ammonium chloride, and detergents such as Triton X-100. These agents arecommercially available. The present invention further contemplates theuse of detergent along with isotonic saline as described by Kim, U.S.Pat. No. 4,099,917, hereby incorporated by reference. The presentinvention further contemplates selective lysis using water as well asselective lysis by freezing and thawing.

The preferred selective lysing agent of the present invention is oneutilizing quaternary ammonium salts. Quaternary ammonium salts canadvantageously be employed as a stromatolysing agent, with the virtuallyinstantaneous destruction of red blood cells but yet without destructionof white blood cells. The quanternary ammonium ion in the salt is of thetype having three short chain alkyl groups and one long chain alkylgroup attached to nitrogen. Referring to the short chain alkyl groups asR₁, R₂, and R₃, these may have from 1 to 4 carbon atoms, as representedby methyl, ethyl, propyl, and butyl. Referring to the long chain alkylgroup as R₄, this may vary in the range of about 10 to 20 carbon atoms(from decyl to eicosyl). For example, trimethyl tetradecyl ammoniumhalides are described by Hamill, U.S. Pat. No. 3,874,852, herebyincorporated by reference. Ledis et al., U.S. Pat. Nos. 4,485,175 and4,286,963, hereby incorporated by reference, also describe a number ofknown selective cell lysing agents which contain quaternary ammoniumhalides. The lyric solutions may contain potassium cyanide, sodiumnitrite or sodium nitroferricyanide. On the other hand, a cyanideion-free lysing agent may used as described by Lancaster et al., U.S.Pat. No. 4,185,964, hereby incorporated by reference. Other quaternaryammonium salt-containing lysis agents may be employed in the selectivelysis step of the present invention. See e.g. Carter et al., U.S. Pat.Nos. 4,346,018 and 4,521,518, Larsen, U.S. Pat. No. 4,521,518, Matsudaet al., U.S. Pat. No. 4,617,275, and Lapicola et al., U.S. Pat. No.4,745,071, hereby incorporated by reference.

Commercial, quaternary ammonium salt-containing lysis agents areavailable from Sequoia-Turner Corporation (Mountain View, Calif.,U.S.A.). These agents lyse red blood cells instantly and break down redcell fragments and their stroma or ghosts. Whole blood may be diluted(1:10 or more) with diluent containing white blood cell stabilizingagents such as 1,3-dimethylurea before treatment with lysing agents. Apreferred commercial selective cell lysis agent for the presentinvention is ZAP-OGLOBIN® manufactured by Coulter Diagnostics, adivision of Coulter Electronics, Inc. (Hialeah, Fla., U.S.A.). WhenZAP-OGLOBIN® is used, it is preferred that it is diluted in ISOTON®II, adiluent manufactured by Coulter Diagnostics and described in U.S. Pat.No. 3,962,125, hereby incorporated by reference.

Cell Concentration

The major concern with red blood cell lysis is the release of heme andrelated compounds which inhibit Taq polymerase. PCR Technology, H. A.Erlich (ed.) Stockton Press (1989) p.33. It can be estimated that theconcentration of heme in hemolyzed whole blood is approximately 10 mMassuming normal hemoglobin concentration in whole blood is 15 g/100 ml.Hematology, 2nd ed., W. J. Williams et al. (ed.) McGraw-Hill, N.Y.(1977). This is far above the reported inhibitory concentration of 0.8μM.

Following the selective lysis step, a process step is performed thatinvolves concentration of the cells. As noted above, concentration isdefined broadly and may result from a wide variety of techniques,including filtration, agglutination, centrifugation, immobilization,and/or fluid evaporation. In a preferred embodiment, the concentrationstep simultaneously removes polymerase inhibitors such as heme andrelated compounds.

In one embodiment, centrifugation at 250 g for 10 minutes is performed,pelleting predominantly white blood cells and leaving the majority ofplatelets in the supernatant. In another embodiment, microfuging for asshort as one minute is performed, pelleting both white blood cells andplatelets. The presence of platelets is found not to impact subsequentsteps of the whole blood processing method of the present invention. Theadvantages of this white blood cell concentration step are: (1)centrifugation is quick (compare, for example, gradient centrifugation);(2) stored blood can be used; 3) cells are in pellet form and thuseasier to collect than cells supported at a gradient interface; and, (4)essentially complete (quantitative) recovery of white blood cells ispossible (i.e. high yield).

The centrifugation step to pellet white blood cells may be varieddepending on which version (a or b, see FIG. 1) of Mode I is utilized.Where version a is utilized, an inhibitor treatment step is employed.Since an inhibitor treatment step will avoid problems with residualpotential polymerase inhibitors, the prior centrifugation step can beperformed with only one centrifugation. With one centrifugation, themajority of the heme is removed with the supernatant and the remainingheme can be successfully treated in the later step. On the other hand,where no inhibitor treatment step is employed, it has been determinedthat two washings of the cell pellet are necessary for the residual hemeto be at non-inhibitory levels.

In another embodiment, filters are used for cell concentration. Indeed,the present invention demonstrates the efficacy of some filters--but notothers--to separate white blood cells from a small volume (less than 1ml and preferably less than 0.1 ml) of whole blood that has beensubjected to selective lysis.

Non-Selective Cell Lysis

Following concentration, the cells can then be non-selectively lysed.The preferred non-selective lysing agent is protease K. Protease K is aproteolytic enzyme from Tritirachium album. It is particularly useful inthe present invention because it has no significant DNAse activity and,therefore, does not render nucleic acid unamplifiable. It is alsoattractive because it is inexpensive and commercially available (e.g.Sigma, St. Louis, Mo., U.S.A., catalogue No. p4914 "Proteinase K").Various treatment conditions using protease K have been found useful. Itis preferred that high concentration of protease K (e.g. 1.5-2.5 mg/ml)be used for short (5-10 minutes) incubation periods to completelydegrade cellular as well as plasma protein and expose cellular nucleicacid for amplification. When lower protease K concentrations (e.g. 0.5mg/ml) are used, longer incubation periods (30-60 minutes) are requiredto achieve the same effect. So that amplification may be carried out, itis preferred that protease K is inactivated. Inactivation may beachieved thermally (preferred) or chemically. If chemical, the preferredinactivation is by the addition of a chelating agent, such asethylenediaminetetraacetic acid (EDTA). EDTA removes Ca⁺² ions requiredfor protease K activity. See Bajorath et al., Nature 337:481 (1989).

Where bacterial cells are believed to be contaminating the white cellpreparation, it may be desirable to lyse the bacterial cells prior to,together with, or after lysis of the white cells. This may depend onwhether amplification of bacterial sequences is intended. Lysis ofbacterial cells can be accomplished enzymatically or chemically. Ifaccomplished enzymatically, it is contemplated that lysozyme ormutanolysin is used, both of which are commercially available (e.g.Sigma). Lysozyme (also called "muramidase") is a single polypeptide of129 amino acids which dissolves bacterial cell wall mucopolysaccharidesby hydrolyis. Mutanolysin apparently exists in two major molecularforms, a 22,000 MW and an 11,000 MW form. Both forms have pronouncedlytic activity on a vast array of bacteria.

Following non-selective lysis, Mode I may or may not have another stepbefore amplification (see FIG. 1). In one version ("a"), the methodproceeds directly to amplification. In another ("b"), the methodproceeds to an inhibitor treatment step.

Inhibitor Treatment

As noted above, by "inhibitor treatment" it is meant that a process iscarried out to treat some potential, residual polymerase inhibitors suchas heme and its related compounds. The process may be chemical,biochemical, photochemical, immunological, or enzymatic, or anycombination thereof. In each case, the inhibitor treatment of thepresent invention does not render the nucleic acid unamplifiable, i.e.the treatment does not impair subsequent amplification.

Chemical

In one embodiment, a chemical process is utilized that is directed atthe chemical breakdown of potential inhibitors by disrupting heme-typeinhibitors by either oxidation or reduction so as to change theirgeometry or coordination chemistry, preventing interaction withpolymerase. Where reduction chemistry is used, the preferred reducingagent is a hydride, such as sodium borohydride. Where oxidation is used,the preferred oxidizing agent is ascorbate.

Biochemical

In one embodiment, a biochemical process is utilized wherein a reagentis introduced that will interfere with the inhibition of polymerase byinhibitors present in whole blood. In one embodiment, such "interferingreagents" comprise compounds selected from the group consisting ofporphyrin-binding compounds. The present invention contemplates thatporphorin-binding compounds include heme-binding compounds, such as the93,000 molecular weight, heme-binding protein from rabbit serum. SeeTsutsui and Mueller, J. Biol. Chem. 257, 3925-3931, 1982). Thisheme-binding protein appears to be unique to rabbits; no analogousproteins have been found in other species tested thus far (human, calfand rat).

In one embodiment, interfering reagents comprise compounds selected fromthe group consisting of globin, serum albumin, and transferrin. Thepreferred compounds are serum albumin and transferrin. Where serumalbumin or transferrin are used to treat polymerase inhibitors, thepresent invention provides cofactors to be used in conjunction.Cofactors are characterized in that they are typically mono- or divalentanions. In one embodiment, the cofactor is selected from the groupconsisting of bicarbonate ion, azide ion, thiocyanate ion, cyanate,oxalate ion, malonate ion, glycinate ion and thioglycolate ion. Thecofactor normally is supplied as a salt (e.g. sodium bicarbonate). Thepreferred cofactors when transferrin is used are bicarbonate ion,thiocyanate ion, cyanate, glycinate ion and thioglycolate ion.

Bovine serum albumin (BSA) has been used in PCR to overcome aninhibitory activity of unknown origin that is present in many extractsof ancient DNA (i.e. museum specimens and archaeological finds). See PCRProtocols: A Guide To Methods and Applications, Innis, M. A. et al.eds., pp. 159-166 (1990). Inhibition of polymerase by heme-typecompounds has not been shown to be overcome with BSA. The presentinvention describes this for the first time and demonstrates a cofactorrequirement.

Transferrin, a blood serum component, is an iron transport protein. Itsmolecular weight is reported to be between 70,000-90,000 depending onthe species of origin. Metal ion binding of transferrin is a wellstudied phenomenon. For example, in the presence of an anion (such asHCO₃ ⁻), transferrin has the capacity to take up 2 iron atoms permolecule. There are, however, no previous reports regarding the abilityof transferrin to overcome the inhibition of polymerases by heme.

While not limited to any mechanisms, it would appear that the hereindescribed ability of transferrin to overcome the inhibition ofpolymerases by heme-type compounds is not mediated by metal ion binding.First there is the question of stoichiometry: the molar ratio oftransferrin to heme at which transferrin is most effective in bindingheme is <0.27. This is lower than that expected from the molar ratio oftransferrin to iron binding ratio of 0.5. Second, there is the questionof the central metal ion: the central metal iron was found not to berequired for the transferrin-mediated phenomenon of the presentinvention. This is evident from the demonstration (described herein)that transferrin is able to overcome the inhibition of polymerases byprotoporphyrin, a heme molecule free of the central metal iron.

Photochemical

The present invention also contemplates a photochemical treatment toeliminate inhibition of polymerase by inhibitors. A preferredphotochemical treatment comprises exposure of potential inhibitors toultraviolet radiation. Particular types of ultraviolet radiation areherein described in terms of wavelength. Wavelength is herein describedin terms of nanometers ("nm"; 10⁻⁹ meters). For purposes herein,ultraviolet radiation extends from approximately 180 nm to 400 nm. Whena radiation source does not emit radiation below a particular wavelength(e.g. 300 nm), it is said to have a "cutoff" at that wavelength (e.g. "awavelength cutoff at 300 nanometers").

When ultraviolet radiation is herein described in terms of irradiance,it is expressed in terms of intensity flux (milliwatts per squarecentimeter or "mW cm⁻² "). "Output" is herein defined to encompass boththe emission of radiation (yes or no; on or off) as well as the level ofirradiance.

A preferred source of ultraviolet radiation is a fluorescent source.Fluorescence is a special case of luminescence. Luminescence involvesthe absorption of electromagnetic radiation by a substance and theconversion of the energy into radiation of a different wavelength. Withfluorescence, the substance that is excited by the electromagneticradiation returns to its ground state by emitting a quantum ofelectromagnetic radiation. While fluorescent sources have heretoforebeen thought to be of too low intensity to be useful for photochemicaltreatment, in one embodiment the present invention employs fluorescentsources to achieve photochemical treatment of polymerase inhibitors. Apreferred fluorescent source is a device ("HRI-100") sold commerciallyby HRI Research Inc. (Berkeley, Calif., USA) and ULTRA-LUM, INC.(Carson, Calif., USA).

Immunological

The present invention also contemplates an immunological treatment toeliminate inhibition of polymerase by inhibitors. Where specificinhibitors are sought to be treated immunologically, the presentinvention contemplates the use of antibodies directed at suchinhibitors. In one embodiment, antibodies are raised by immunizationwith specific inhibitors. The inhibitors may be obtained from cellsdisrupted by treatments which include sonic disruption, osmotic changeor use of agents as organic solvents, detergents, enzymes and the like.

Furthermore, immunological equivalents of the inhibitors (e.g. proteinfragments) may be used to facilitate the production of antibodies.Additionally, a mixture of inhibitors may be used to facilitate theproduction of antibodies.

The antibodies may be monoclonal or polyclonal. It is within the scopeof this invention to include any second antibodies (monoclonal orpolyclonal) directed to the first antibodies discussed above. Themethods of obtaining both types are well known in the art. Polyclonalsera are relatively easily prepared by injection of a suitablelaboratory animal with an effective amount of the inhibitor or antigenicparts thereof, collecting serum from the animal, and isolating specificsera by any of the known immunoadsorbent techniques.

The preparation of hybridoma cell lines for monoclonal antibodyproduction derived by fusing an immortal cell line and lymphocytessensitized against the immunogenic preparation can be done by techniqueswhich are well known to those who are skilled in the art. (See, forexample Douillard and Hoffman, Basic Facts about Hybridomas, inCompendium of Immunology Vol II, ed. by Schwartz, 1981; Kohler andMilstein, Nature 256: 495-499, 1975; European Journal of Immunology 6:511-519, 1976).

Unlike preparation of polyclonal sera, the choice of animal is dependenton the availability of appropriate immortal lines capable of fusing withlymphocytes. Mouse and rat have been the animals of choice in hybridomatechnology and are preferably used.

The utilization of an antibody produced in the above-described mannermay be accomplished in a number of ways such as providing antibody on asolid support and passing the products of the non-selective lysis stepover the solid support. On the other hand, the antibody may beintroduced in solution to the products of the non-selective lysis step.

Enzymatic

The present invention also contemplates an enzymatic treatment toeliminate inhibition of polymerase by inhibitors. For example, thepresent invention contemplates the use of heme oxygenase to treatheme-type inhibitors. Heme oxygenase is the enzyme associated with thefirst step of heme catabolism in eukaryotes. Degradation of heme to bilepigments is mediated by heme oxygenase. Molecular oxygen (as well asNADPH) is required for this oxidation which results in attack at thealpha meso carbon bridge of the heme, releasing CO and formingbiliverdin. See Schacter, Sem. in Hem. 25:349 (1988). Thereafter,biliverdin may be converted to bilirubin by the enzyme biliverdinreductase.

Combination

The present invention also contemplates an inhibitor treatment step thatinvolves a combination of the above-named process steps. For example,the present invention contemplates the chemical oxidation of heme-typeinhibitors followed by photochemical destruction of the products ofchemical oxidation. In another embodiment, the present inventioncontemplates the enzymatic degradation of heme-type inhibitors followedby the photochemical destruction of the products of enzymaticdegradation. In still another embodiment, the present inventioncontemplates an inhibitor treatment step that involves a simultaneoususe of two or more of the above-named process steps (e.g. chemicalreduction in the presence of particular ultraviolet wavelengths).

Mode II

Cell Concentration

As noted above, "concentration" is defined broadly and may result from awide variety of techniques, including immobilization and/or fluidevaporation. In one embodiment, the present invention contemplatesimmobilizing whole blood in low melting agarose. In this embodiment, theblood sample is placed in warm (melted) agarose which is then congealedat room temperature. The agarose can thereafter be melted for subsequentprocessing steps (see below).

In another embodiment, whole blood is immobilized on an absorptivesupport and dried. In this regard, the present invention demonstratesthe efficacy of some filters--but not others--to immobilize blood cellsfrom a small volume (less than 1 ml and preferably less than 0.1 ml) ofwhole blood.

In another embodiment, white blood cells are immobilized on a solidsupport while red blood cells are not. With respect to the latter,various filters have been used and proven effective in immobilizing onlywhite blood cells.

Since platelets are much smaller than either red blood cells or whiteblood cells, and red blood cells have a different shape from white bloodcells, pore sizes can be chosen to allow passage of platelets and redblood cells while facilitating adhesion of white blood cells to thefilter materials. Different pore sizes and filtration flow rates havebeen found useful in the concentration step of the present invention.

Non-Selective Cell Lysis

As noted for Mode I, the preferred non-selective lysing agent for ModeII is protease K. Other non-selective lysis approaches, however, arealso contemplated, including lysis by heating whole blood. Where wholeblood has been previously immobilized by adding to a filter and drying,the present invention contemplates non-selective cell lysis in thepresence of the filter. In one embodiment, the filter is selected thatwill subsequently dissolve during the non-selective lysis step (e.g.with protease K, a protein filter is selected). In another embodiment,the filter disc is impregnated with the lysing agent prior toimmobilization of whole blood. In this way, the concentration step andthe lysis step of Mode II are performed simultaneously.

Inhibitor Treatment

The inhibitor treatment for Mode II is also contemplated to be chemical,biochemical, photochemical, immunological, or enzymatic, or anycombination thereof. Where whole blood has been previously immobilizedby adding to a filter and drying, the present invention contemplatesinhibitor treatment in the presence of the filter. In one embodiment,the filter is selected that will be tolerated by the inhibitor treatment(e.g. with a chemical inhibitor treatment, an inert filter is selected).In another embodiment, a second filter disc (impregnated with ainhibitor treatment agent such as interfering reagent) is used alongwith the filter disc having the immobilized whole blood. In this way,the concentration step and the lysis step of Mode II are performedsimultaneously on one disc and the inhibitor treatment is performedthereafter by the introduction of a second disc.

Mode III

Non-Selective Cell Lysis

As noted for Mode I, the preferred non-selective lysing agent for ModeII is protease K. Other non-selective lysis approaches, however, arealso contemplated, including lysis by heating whole blood.

Inhibitor Treatment

The inhibitor treatment for Mode II is also contemplated to be chemical,biochemical, photochemical, immunological, or enzymatic, or anycombination thereof.

From the above, it is clear that the present invention provides auseful, and yet flexible, approach to preparing nucleic acid sequences.Which mode is most appropriate depends on the concentration of thesequence of interest. The concentration of the sequence of interest isdetermined by the sample size and copy number. For low copy numbersystems, one typically needs a larger sample size in order to be surethe sequence of interest is present in the sample. Large sample sizes,however, have more inhibitor. Furthermore, large samples are not readilyamenable to amplification because of the expense of large sampleamplification as well as the inhibiting impact of vary large amounts ofnucleic acid on most amplification techniques.

The above considerations are best understood by example. The low copysituation of virus infection is a good case in point. Since the numberof HIV infected T4 cells can be as low as one out of every 10,000 andonly 14% of the total white blood cell population is T4, for each HIVPCR beginning with 40 infected T4 cells, a minimum of 300-600 μl ofnormal whole blood will be required. A larger volume may be needed forHIV patients due to depletion of their T4 cells. However, since the PCRis normally carried out in 100 ul, a volume reduction step, which allowsconcentrations of white blood cells, may be necessary to avoid theexpense of large amounts of enzyme.

One potential problem of the cell concentration steps is that the finalamount of DNA obtained by the procedure may be too high for the PCR toefficiently proceed. It has been shown that the amount of DNA present in0.5 ml of normal whole blood is difficult or impossible to amplify allat once by the PCR. The occurrence of DNA dependent PCR inhibition isprobably due to an excess of misprimed sites (relative to enzymemolecules), which form unproductive ternary complexes with thepolymerase. This results in the accumulation of a large number oflinearly or exponentially amplified non-target sequences. Since thespecificity of the amplification is lost as the amount of non-target DNAis increasing, the exponential accumulation of the target sequence ofinterest does not occur to any significant degree.

EXPERIMENTAL

In the experimental disclosure which follows, all weights are given ingrams (g), milligrams (mg) or micrograms (μg), all lengths are given ascentimeters (cm), millimeters (mm), micrometers (μm) or nanometers (nm),all pressures are given as pounds per square inch (psi), alltemperatures are given as degrees Centigrade (°C.), all concentrationsare given as percent by weight (% or percent), equivalents (eq), Normal(N), molar (M), millimolar (mM) or micromolar (μM), all quantities aregiven as moles (mol), millimoles (mmol), micromoles (μmol) or nanomoles(nmol) and all volumes are given as liters (l), milliliters (ml), ormicroliters (μl) unless otherwise indicated.

In addition, the following abbreviations have the indicated meanings: MW(molecular weight); OD (optical density); EDTA(ethylenediaminetetraacetic acid); TE buffer (buffer: 10 mM Tris/1 mMEDTA, pH 7.5); TBE buffer (Tris-Borate-EDTA); TAE buffer(Tris-Acetate-EDTA); Taq buffer (50 mM KCl, 2.5 mM MgCl₂, 10 mM Tris, pH8.5, 200 μg/ml gelatin); PAGE (polyacrylamide gel electrophoresis);SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); V(volts); W (watts); mA (milliamps); bp (base pair); kb (kilobase pairs);Ci (Curies); μCi (microcuries); rxn (reaction) and CPM (counts perminute).

Generally, PCR was carried out using 175-200 μM dNTPs(deoxyribonucleoside 5'-triphosphates) and 0.5 to 1.0 μM primers. 5Units/100 μl of Taq polymerase was used. PCR reactions were overlaidwith 30-100 μl light mineral oil. A typical PCR cycle for HIVamplification using a Perkin-Elmer Cetus DNA Thermal Cycler (Part No.N8010150) was: denaturation at 95° C. for 30 seconds; annealing at 55°C. for 30 seconds; and extension at 72° C. for 1 minute. PCR cycles werenormally carried out in this manner for 35 cycles followed by 7 minutesat 72° C.

In some instances below, amplification of human histocompatibility (HLA)Class II genes was performed using primer pair GH26/GH27 and humanplacental DNA to produce a 242-mer product. The sequences of theseprimers are:

    GH26 5'-GTGCTGCAGGTGTAAACTTGTACCAG-3'                      (SEQ ID NO:2)

    GH27 5'-CACGGATCCGGTAGCAGCGGTAGAGTTG-3'                    (SEQ ID NO:3)

These primers and other primers are described in PCR Protocols: A GuideTo Methods and Applications, Innis, M. A. et al. eds., pp. 261-271(1990)). The sequence of the 242-mer product is:

    ______________________________________                                        242-  5'-GTGCTGCAGG TGTAAACTTG TACCAGTTTT                                     mer   ACGGTCCCTC TGGCCAGTAC ACCCATGAAT                                              TTGATGGAGA TGAGGAGTTC TACGTGGACC                                              TGGACAGGAA GGAGACTGCC TGGCGGTGGC                                              CTGAGTTCAG CAAATTTGGA GGTTTTGACC                                              CGCAGGGTGC ACTGAGAAAC ATGGCTGTGG                                              CAAAACACAA CTTGAACATC ATGATTAAAC                                              GCTACAACTC TACCGCTGCT ACCGGATCCG                                              TG-3' (SEQ ID NO: 4)                                                    ______________________________________                                    

In other cases, amplification of HIV sequences was performed usingprimer pair SK-38/SK-39 and 115-mer as target to produce a 115-merproduct. The sequences of these primers are:

    ______________________________________                                        SK38   5'-ATAATCCACCTATCCCAGTAGGAGAAAT                                               (SEQ ID NO: 5)                                                         SK39   5'-TTTGGTCCTTGTCTTATGTCCAGAATGC                                               (SEQ ID NO: 6)                                                         ______________________________________                                    

The sequence of the 115-mer product is:

    ______________________________________                                        115-  5'-ATAATCCACC TATCCCAGTA GGAGAAATTT                                     mer   ATAAAAGATG GATAATCCTG GGATTAAATA                                              AAATAGTAAG AATGTATAGC CCTACCAGCA                                              TTCTGGACAT AAGACAAGGA CCAAA-'3                                                (SEQ ID NO: 7)                                                          ______________________________________                                    

In other cases, amplification of HCV sequences was performed usingprimers having the following sequences:

    ______________________________________                                        KY78  5'-XCTCGCAAGCACCCTATCAGGCAGT (25-mer)                                         (SEQ ID NO: 8)                                                          KY80  5'-GCAGAAAGCGTCTAGCCATGGCGT (24-mer)                                          (SEQ ID NO: 9)                                                          ______________________________________                                    

In one case, amplification of globin sequences was performed usingprimer pair KM-29/HRI-12. These primers give a 174 bp product within thehuman beta globin gene. The sequences of these primers are:

    ______________________________________                                        KM-29     5'-GGTTGGCCAATCTACTCCCAGG-3'                                                  (SEQ ID NO: 10)                                                     HRI-12    5'-GGCAGTAACGGCAGACTACT-3'                                                    (SEQ ID NO: 11)                                                     ______________________________________                                    

Where agarose gel electrophoresis was used, the PCR products werefractionated by electrophoresis through a 3% Nusieve/1% agarose gel inTBE buffer. Electrophoresis was typically carried out in a constantvoltage of 8-10 V/cm. Following agarose gel electrophoresis, individualbands were, in most cases, visualized by ethidium bromide staining. Thisinvolved staining the agarose gel with 1 μg/ml ethidium bromide in TBEfor 30 minutes followed by destaining the agarose gel with TBE for 30minutes.

Where polyacrylamide gel electrophoresis (PAGE) was used, two types ofgels were used, denaturing and native. Denaturing (7 or 8M urea)polyacrylamide gels (28 cm×35 cm×0.4 mm) were poured andpre-electrophoresed for 30 to 60 minutes at 2000 Volts, 50 Watts, 25milliamps. 12% gels were used for oligonucleotides between 40 and 200base pairs in length; 8% gels were used for longer sequences. Dependingon the length of DNA to be analyzed, samples were loaded in either 8Murea, containing 0.025% tracking dyes (bromphenol blue and xylenecyanol), or in 80% formamide, 10% glycerol, 0.025% tracking dyes, thenelectrophoresed for 2-4 hours at 2000 Volts, 50 Watts, 25 milliamps.Following PAGE, individual bands were, in most cases, visualized byautoradiography. Autoradiography involved exposure overnight at -70° C.to Kodak XAR-5 films with an intensifying screen.

In order to visualize with autoradiography, PCR products were internallyradiolabelled. This simply involved adding 2 μCi of α-³² P-dCTP (3000Ci/mmole, NEN Research Products, Boston Mass., U.S.A.) to each PCRreaction. The internally-radiolabelled PCR products are directlyfractionated by this denaturing PAGE without prior treatment.

Native polyacrylamide gels were poured similarly to the denaturing gelwith the urea left out. No pre-electrophoresis was necessary. Sampleswere loaded in 10 mM Tris, 10 mM EDTA, 0.1% SDS, 10% glycol, and 0.025%tracking dyes. Following PAGE, analysis was carried out as describedabove for denaturing gels.

SDS-PAGE was used to fractionate protein molecules. Generally, the gelused was 10% polyacrylamide in 0.375M Tris pH 8.8 and 0.1% SDS. Thestacking gel was 5% polyacrylamide in 0.125M Tris pH 6.8 and 0.2% SDS.Running buffer was 0.025M Tris pH 8.3, 0.192M glycine and 0.1% SDS.Protein samples were dissolved in 0.0625M Tris pH 6.8, 2% SDS, 5%β-mercaptoethanol containing 10% glycerol and tracking dye (BPB) andheated to 100° C. for 2 minutes before loading. After electrophoresis,the protein bands were visualized after staining with 0.1% Coomassieblue (Sigma) in 50% Methanol and 10% trichloroacetic acid for 1 hourfollowed by destaining in 5% methanol and 7% acetic acid.

Ouchterlony immunodiffusion gels were used. See generally, E. A. Kabat,Structural Concepts in Immunology and Immunochemistry (Holt, Rinehartand Winston, N.Y. 1968). An agarose solution is poured into a standardpetri dish. After the gel hardens, a center well and surrounding wellsare punched out and the agarose plugs removed. A solution of antibody isplaced in the center well and solutions of the relevant test samples areplaced separately in the surrounding wells. The petri dish is placed at4° C. overnight and then inspected visually for precipitin lines.

Photochemistry was performed using a device ("HRI-100") soldcommercially by HRI Research Inc. (Berkeley, Calif., USA) and ULTRA-LUM,INC. (Carson, Calif., USA).

In some cases, amplified products were visualized by OligonucleotideHybridization (OH) analysis. PCR products were mixed with radiolabeledprobe in 10 mM EDTA, 15 mM NaCl, denatured for 5 minutes at 95° C.,followed by an annealing step at 55° C. for 15 minutes. The hybrids wereseparated from unincorporated probe by PAGE with a native gel.

Probes were end-labeled with γ-³² P-ATP by T₄ polynucleotide kinase.Typically, 0.2 μg of an oligonucleotide probe was incubated with 20 μCiγ-³² P-ATP (6000 Ci/mmole, NEN Research Products, Boston Mass., U.S.A.)and 20 units of T₄ polynucleotide Kinase (New England BioLab) at 37° C.for one hour. After stopping the reaction with 25 mM EDTA, the labeledprobe was separated from unincorporated γ-³² P-ATP by a spin columnchromatography. A mini-Sephadex G-50 column (1 ml) was packed bycentrifugation (2 min, 1,800 rpm in a table top centrifuge) in a 1 mldisposable syringe in TE. The T₄ Kinase reaction products were loadedover the top of the column and centrifuged again at 1,800 rpm for 2minutes. The labeled probe is collected in the exclusion volume.

In some examples, clinical samples were supplied by the CaliforniaDepartment of Public Health (CDPH) and used to validate the compoundsand methods of the present invention. Each of the high risk patientsamples were evaluated by the CDPH for antibody to HIV ("serotested") byELISA assay using the Organon Teknika ELISA kit. This kit containsmultiple antigens with predominantly p24. Each sample was tested twice.

The ELISA results were thereafter confirmed by an immunofluorescenceassay (IFA) performed at the CDPH. For this test, HIV infected H9 cellsexpressing HIV antigens were fixed on microscope slides. A drop of serumfrom the clinical sample was added to the fixed cells. After incubation,unbound serum constituents were washed away. A second antibody specificto human immunoglobin conjugated with fluorescein isothiocyanate (FITC)was added. The bond serum antibody to HIV was then visualized under afluorescent microscope.

In case there is a disagreement between the ELISA results and the IFAresults, then both the Western Blot and Radioimmunoprecipitation Assay(RIPA) were used to determine if a sample was indeed positive.

The following examples are provided in order to demonstrate and furtherilluminate certain aspects of the practice of the invention.

Example 1

In this example, the effect of chemical treatment of hematin oninhibition of amplification is demonstrated. The chemical reaction wasperformed as follows. Two aliquots (30 mg/aliquot) of sodium borohydride(NaBH₄) were added at ten minute intervals to 1 ml of 200 μM hematin(Sigma) in 1M Tris (pH 8.0) under argon in the dark. The pH was adjustedto 6.0 between additions. At the end of the reaction (20 minutes) the pHwas adjusted to 1.0 and then neutralized to pH 7.0. Six aliquots (5 μl)of the product of the reaction corresponding to different amounts ofreacted hematin (37.5 μM, 7.5 μM, 1.5 μM, 0.3 μM, 0.06 μM, 0.012 μM)were added to standard (20 μl) PCR reactions in different tubes usingthe SK38/39 system (see above). Three standard control reactions wererun in separate tubes: i) a no nucleic acid target control, ii) a noprimer control, and iii) a positive control (no hematin). In addition,unreacted hematin was added to four different tubes in different amounts(50 μM, 5 μM, 0.5 μM, and 0.05 μM). PCR was carried out for 30 cycles.The PCR products were thereafter electrophoresed (agarose) andvisualized with ethidium bromide staining (FIG. 2A, lanes 1-13). Thethree above-named control reactions were electrophoresed in lanes 1, 2and 3, respectively. As expected, no product is visible in lanes 1 and 2(negative controls), while a strong product band corresponding to theexpected 115-mer is apparent in lane 3 (positive control). The unreactedhematin tubes (lanes 4-7) show no product until hematin is present at avery low concentration (0.05 μM, lane 7), at which point product isevident. On the other hand, product is visible in some of the lanes(lanes 10-13) representing reactions where treated hematin was added(lanes 8-13). Indeed, product is evident where 1.5 μM reacted hematin orless was present. FIG. 2B shows a more quantitative analysis of thereaction based on the best visual estimation of the band intensity. Ascore (i.e. % maximum intensity of a positve control) was given to eachband. An OD unit of 1.0 corresponds to 100% of maximum intensity. Afterexamination of a plot of the estimated OD units to the concentration ofhematin (expressed in the log scale) in these PCR reactions, it is clearthat approximately a thirty-fold concentration difference was achieved(reacted to unreacted).

Example 2

In this example, the effect of photochemical treatment of biliverdin oninhibition of amplification is demonstrated. The photochemical reactionwas performed as follows. Stock biliverdin (Sigma, 5 mM) was diluted insolution (50 mM Tris pH 7.8) to a final concentration of 200 μM. Twoaliquots (300 μl) were then made. One aliquot was left unreacted. Theother aliquot was exposed to ultraviolet radiation from an HRI-100 for16 hours using the industry standard, F8T5BL hot cathode dual bipinlamps. Thereafter, both the unirradiated and irradiated materials wereadded (5 μl) to standard (20 μl) PCR reactions in different tubes usingthe SK38/39 system (see above) and 10⁹ copies of 115-mer target. Threestandard control reactions were run in separate tubes: i) a no nucleicacid target control, ii) a positive control (no biliverdin), and iii) ano Taq control. PCR was carried out for thirty (30) cycles. The PCRproducts were thereafter electrophoresed (agarose) and visualized withethidium bromide staining (FIG. 3A, lanes 1-5). The three above-namedcontrol reactions were electrophoresed in lanes 1, 2 and 3,respectively. As expected, no product is visible in lanes 1 and 3(negative controls), while a strong product band corresponding to theexpected 115-mer is apparent in lane 2 (positive control). Theunirradiated biliverdin tube (lanes 4) shows no product, (i.e., PCR iscompletely inhibited). On the other hand, product is visible in the lane(lane 5) representing the reaction where photochemically treated (16hrs) biliverdin was added.

A spectroscopic analysis of the photochemical treatment is shown in FIG.3B. In this case 0.5 ml of untreated biliverdin in solution (diluted in50 mM Tris pH 7.8) to a final concentration corresponding to A_(peak)=0.7) was scanned using a Beckman Spectrophotometer Model DU-50 between300 and 700 nm. Photochemically treated (1.5 hours and 16 hours ofirradiation) biliverdin was similarly diluted and scanned. The resultsare shown in FIG. 3B. The untreated biliverdin shows a strong absorbancepeak at approximately 450 nm. With just 1.5 hours of irradiation, thispeak is very weak; with 16 hours, it is eliminated completely. Thiscorresponds to lane 5 in FIG. 3A where no PCR inhibition was observed.

Example 3

In this example, the effect of photochemical treatment of bilirubin oninhibition of amplification is demonstrated. The photochemical reactionwas performed as follows. Stock bilirubin (Sigma, 5 mM) was diluted insolution (50 mM Tris pH 7.8)) to final concentrations of 400 μM, 800 μMand 2 mM. Two aliquots (300 μl) at each concentration were then made.One aliquot of each concentration was left unreacted. The other aliquotwas exposed to ultraviolet radiation from an HRI-100 for 0-4 hours usingthe industry standard, F8T5BL hot cathode dual bipin lamps. Thereafter,both the unirradiated and irradiated material were added (5 μl) tostandard (20 μl) PCR reactions in different tubes using the SK38/39system (see above) and 10¹¹ copies of 115-mer target. Six standardcontrol reactions were run in separate tubes: i) a no nucleic acidtarget control, and ii-vi) five positive controls (no bilirubin), eachstarting with a different copy number (10¹¹, 10⁹, 10⁷, 10⁵, and 10³).PCR was carried out for thirty (30) cycles in the presence of α-³²P-dCTP (NEN Research Products, Boston Mass., U.S.A.). The PCR productswere thereafter electrophoresed (denaturing PAGE), and visualized byautoradiography (FIG. 4A, lanes 1-12). The six above-named controlreactions were electrophoresed in lanes 1-6, respectively. As expected,no product is visible in lane 1, while a product band corresponding tothe expected 115-mer is apparent in lanes 2-6. Where unreacted bilirubinwas used (lanes 7, 9 and 11) there is no product evident, i.e. PCR iscompletely inhibited. On the other hand, product is visible in the lanes(lane 8, 400 μM bilirubin, 2.5 hrs; and lane 10, 800 μM bilirubin, 3.5hrs) representing the reaction where photochemically treated bilirubinwas added in concentrations less than 800 μM. Product is not visible inthe lane (lane 12, 2 mM bilirubin, 4 hrs) representing the reactionwhere photochemically treated bilirubin was added in very highconcentration (2 mM).

A spectroscopic analysis of the photochemical treatment is shown in FIG.4B. In this case 0.5 ml of untreated bilirubin in solution (diluted in50 mM Tris, pH 7.8 to a final concentration corresponding to A_(peak)=0.8) was scanned using a Beckman Spectrophotometer Model DU-50 between300 and 700 nm. Photochemically treated (20 minutes, 60 minutes and 2.5hours of irradiation) bilirubin was similarly diluted and scanned. Theresults are shown in FIG. 4B. The untreated bilirubin shows a strongabsorbance peak at approximately 450 nm. With just 20 minutes ofirradiation, this peak is reduced; with just 60 minutes of irradiation,this peak is weak; with 2.5 hours, it is eliminated. The 2.5 hourtreatment corresponds to lane 8 in FIG. 4A where no PCR inhibition wasobserved.

Example 4

In this example, the effect of enzymatic treatment of heme on inhibitionof amplification is demonstrated. The reaction scheme is shown in FIG.5. The enzymatic reaction is performed as follows.

Purification of Heme oxygenase

Heme oxygenase is obtained from bovine spleen (T. Yoshinaga et al., J.Biol. Chem. 257:7778 (1982)), rat liver (R. K. Kutty and M. D. Maines J.Biol. Chem 257:9944 (1982)) or pig spleen or liver (T. Yoshida et al.,J. Biochem 75:1187 (1974). The preferred source is pig and purificationis as follows. Pig spleen or liver (about 1 kg) is homogenized in aWaring Blender in 4 volumes of 0.134M KCl containing 0.02M potassiumphosphate buffer (pH 7.4), and the homogenate is centrifugedsuccessively for 15 min at 8,000×g, for 20 min at 18,000×g, and for 2 hat 56,000×g. The precipitates obtained are washed with 1.6 liters of 1MKCl containing 20 mM potassium phosphate buffer (pH 7.4) and 10 mM EDTAby 2 h of centrifugation at 56,000×g. The resulting precipitates aresuspended in 50 mM potassium phosphate buffer (pH 7.4) containing 10 mMEDTA to give a protein concentration of about 10 mg/ml. To thissuspension is added sodium cholate (20% solution) at a ratio of 0.05 mgof sodium cholate to 1 mg of protein. After stirring for 20 min, thesuspension is centrifuged for 90 min at 77,000×g. The precipitates areresuspended in 50 mM potassium phosphate buffer (pH 7.4) containing 1 mMEDTA to give a protein concentration of 10 mg/ml (the "microsome"fraction). The temperature is maintained at 0°-4° C. through this andother purification procedures. From 1 kg (wet weight) of pig spleen andpig liver, approximately 5 to 7 g and 8 to 10 g are obtained asmicrosomal protein, respectively. Further purification of heme oxygenaseis usually started with 1,500 to 2,000 mg of pig spleen microsomes asprotein which shows the specific activities of heme oxygenase in therange of 13.5 to 32.2 units/mg of protein. Heme oxygenase is solubilizedfrom the microsomes by the addition of a 20% sodium cholate solution ata ratio of 1 mg of sodium cholate to 1 mg of protein. After stirring for60 min, the suspension is centrifuged at 77,000×g for 90 min, and to thesupernatant fluid (cholate fraction) is added solid ammonium sulfate upto 0.4 saturation. The precipitates collected by centrifugation aredissolved in 10 mM potassium phosphate buffer (pH 7.4), containing 1 mMEDTA, 0.1% Triton X-100, and 0.1% sodium cholate, so as to be twice theinitial volume of the cholate fraction. Then the solution is applied toa column of DEAE-cellulose (DE23, 3×30 cm) previously washed with 10 mMpotassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 0.1% TritonX-100, and 0.1% sodium cholate, and the column is washed with about 200ml of 10 mM potassium phosphate buffer (pH 7.4) containing 70 mM KCl, 1mM EDTA, 0.1% Triton X-100, and 0.1% sodium cholate. Heme oxygenase iseluted at a flow rate of 50 ml/h with 600 ml of a similar solution butincreasing the KCl concentration linearly up to 310 mM, and 12-mlfractions are collected. The active heme oxygenase fractions (firstDEAE-cellulose fraction) are combined and diluted 2.5-fold with colddistilled water and applied to a second DEAE-cellulose column (DE32,2.6×28 cm) previously washed with 10 mM potassium phosphate buffer (pH7.4) containing 1 mM EDTA, 0.1% Triton X-100, and 0.1% sodium cholate.After washing the column with 100 ml of the same buffer, heme oxygenaseis eluted with 400 ml of a similar solution using a linear gradientbetween 0 and 200 mM in KCl, at a flow rate of 30 ml/h; 10-ml fractionsare collected. The active fractions (second DEAE-cellulose fraction) arediluted 3-fold with cold distilled water and applied to ahydroxylapatite column (2.6×7 cm) previously washed with 10 mM potassiumphosphate buffer (pH 7.4) containing 1 mM EDTA. The column issuccessively washed with 30 ml of 10 mM potassium phosphate buffer (pH7.4) containing 1 mM EDTA, 50 ml of 200 mM potassium phosphate buffer(pH 7.4) containing 1 mM EDTA, 30 ml of 10 mM potassium phosphate buffer(pH 7.4) containing 1 mM EDTA, and 50 ml of 40 mM potassium phosphatebuffer (pH 7.4) containing 1 mM EDTA, 0.1% Triton X-100, and 0.1% sodiumcholate, at a flow rate of 50 ml/h. Elution of heme oxygenase from thecolumn is performed with 100 ml of 110 mM potassium phosphate buffer (pH7.4) containing 1 mM EDTA, 0.1% Triton X-100, and 0.1% sodium cholate ata flow rate of 30 ml/h; 7-ml fractions are collected. The active hemeoxygenase fractions (hydroxylapatite fraction) are then applied to acolumn of Sephadex G-200 (5×90 cm) previously equilibrated with 30 mMpotassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 0.1% TritonX-100, and 0.1% sodium cholate. Heme oxygenase is eluted with the sameequilibration buffer at a flow rate of 30 ml/h; 15-ml fractions arecollected (Sephadex G-200 fraction).

When the Sephadex G-200 fraction is subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis, only a single protein bandshowing an apparent molecular weight of 32,000 is observed. Thepurification reproducibly yields heme oxygenase preparations having aspecific activity of about 5,200 to 5,650 units with an overall yield of10 to 20%.

Purification of Biliverdin Reductase

The reductase is obtained from bovine spleen according to the protocoldescribed by T. Yoshinaga et al., J. Biol. Chem. 257:7778 (1982). Freshrefrigerated bovine spleens (approximately 1 kg for each purification)are sliced and homogenized in a Waring blender with 4 volumes of 0.25Msucrose in 20 mM Tris-HCl, pH 7.4, containing 5 mM EDTA and 0.5 mMphenylmethylsulfonyl fluoride to inhibit proteolysis. Homogenates arecentrifuged at 15,000×g for 20 min. Precipitates from the 15,000×gcentrifugation are homogenized again with 2 volumes of the sucrosesolution and centrifuged at 15,000×g for 30 min. Supernatants thusobtained are centrifuged at 110,000×g for 60 minutes in a Beckman 60 Tior a 50.2 Ti motor. Supernatant from the 110,000×g centrifugation ofbovine spleen is fractionated from 35% to 65% saturation of ammoniumsulfate, followed by column chromatography on DEAE-cellulose. TheDEAE-cellulose fractions containing major enzyme activity are combinedand concentrated and passed through a column of Sephacryl S-200. Furtherpurification of biliverdin reductase is carried out by affinitychromatography on Sepharose coupled with 2',5'-ADP, a structuralanalogue of NADPH. The preparation obtained is colorless and typicallywill have a specific activity of at least 1100 units/mg of protein,showing a few minor protein bands when analyzed by SDS-polyacrylamidegel electrophoresis.

Inhibitor treatment

Enzymatic inhibitor treatment is performed with hemin. Heme oxygenaseactivity is determined by measuring the bilirubin formation on the basisof increase in absorbance at 468 nm. Standard reaction mixtures containin a final volume of 2 ml: 200 μmol of potassium phosphate buffer (pH7.4), 30 nmol of hemin, 0.2 mg of bovine serum albumin, excess amountsof a partially purified biliverdin reductase, 1 μmol of NADPH,appropriate amounts of NADPH-cytochrome c reductase partially purifiedfrom pig liver microsomes, and heme oxygenase preparation. The last twoenzyme preparations contained 1 mM EDTA, 0.1% Triton X-100, and 0.1%sodium cholate, and the final concentrations of cholate and Triton X-100in the incubation mixture were approximately 0.05%. NADPH is omitted inthe control. The reaction is carried out for 5 to 10 min in a test tubeplaced in a shaking water bath at 37° C. The reaction is started by theaddition of NADPH after a 2-min preincubation and stopped by immersingthe tube in ice water. A value of 43.5 mM⁻¹ cm⁻¹ may be adopted as theextinction coefficient of bilirubin at 468 nm under these conditions.One unit of the enzyme is defined as the amount of enzyme catalyzing theformation of 1 nmol of bilirubin/h under the conditions described above.

Example 6

As noted above, the present invention also contemplates an inhibitortreatment step that involves a combination of the above-named processsteps. In this example, the inhibitor treatment comprises a processwherein both enzymatic and photochemical processes are employed incombination according to the present invention.

FIG. 6 schematically shows inhibitor treatment wherein both enzymaticand photochemical processes are employed in combination according to thepresent invention. In the first part of the scheme, heme-type inhibitorsare treated with heme oxygenase and/or biliverdin reductase according tothe protocol described in Example 4, above. Thereafter, the resultingproduct, biliverdin or bilirubin, is photochemically treated accordingto the protocol described in Example 2 and 3, above.

Example 7

In this example, the inhibitor treatment comprises a process whereinboth chemical and photochemical processes are employed in combinationaccording to the present invention.

FIG. 7 schematically shows inhibitor treatment involving simultaneoususe of chemical oxidation in the presence of ultraviolet radiation. Thechemical oxidation involved the use of ascorbate. Heme undergoes coupledoxidation with ascorbate in the presence of a small quantity ofapomyoglobin. Exposure of apomyoglobin to ascorbic acid and oxygenresults in oxidative cleavage of the ring tetrapyrrole. The finalproduct of heme is biliverdin. Biliverdin can thereafter be inactivatedphotochemically according to the protocol described in Example 2, above.

The reaction was performed as follows. Hemolyzed plasma was obtained byclearing hemolyzed blood by centrifugation at 12,000 rpm for 5 minutesin a microfuge. The total heme concentration was estimated to be 10 mM.Aliquots of "heme" solutions (0, 500 μM) were made in 0.1M sodiumphosphate buffer (pH 7.0) containing 8.4 mM ascorbate and with orwithout 82 μM apomyoglobin. Stock ascorbate (Sigma, 100 mg/ml) wassolubilized by adding 0.1N NaOH to pH 5.0. Tubes were incubated at 37°C. for 5 hours. Thereafter, half of the sample in each ascorbate tubewas added to new tubes and these new tubes were treated photochemicallyusing an HRI-100 for 4 hours using the industry standard, F8T5BL hotcathode dual bipin lamps.

Following irradiation of the new tubes (corresponding to final hemeconcentrations of 125 μM, 25 μM, 5 μM, and 1 μM), aliquots from all thetubes were added to standard (20 μl) PCR reactions in different tubesusing the SK38/39 system (see above) and 10⁹ copies of 115-mer target.Standard control reactions were run in separate tubes. PCR was carriedout for thirty (30) cycles. The PCR products were thereafterelectrophoresed (agarose) and visualized with ethidium bromide staining.As expected, no product was visible with negative controls, while aproduct band corresponding to the expected 115-mer was apparent with thepositive control (data not shown). The lanes corresponding to hemolyzedplasma tubes receiving no ascorbate showed no product, i.e. PCR wascompletely inhibited (data not shown). On the other hand, product wasvisible in all the lanes representing the reactions where ascorbate wasadded (except for the lanes corresponding to the very high concentrationof heme, i.e. 125 μM). When the ascorbate tubes with no photochemicaltreatment were compared with those receiving irradiation, a stronger PCRproduct band was observed in those receiving irradiation.

A quantitative analysis of the results is shown in FIG. 8. The graph isbased on the best visual estimation of the band intensity. A score (i.e.% maximum intensity of a positive control) was given to each band. An ODof 1.0 corresponds to 100% of maximum intensity. After examination of aplot of the estimated OD units to the concentration of inhibitor(expressed in the log scale) in these PCR reactions, it is clear thatapproximately a thirty-fold reduction in inhibition was achieved withthe combination of chemical oxidation and photochemical treatment.

Example 8

In this example, the inhibitor treatment comprises an immunologicalprocess involving the preparation and use of monoclonal antibodies withreactivity for polymerase inhibitors. Mice may be injected with anantigenic amount, for example, from about 0.1 mg to about 20 mg of theinhibitor or antigenic parts thereof. Usually the injecting material isemulsified in Freund's complete adjuvant prior to injection. Boostinginjections may also be required. A crude screen for antibody productioncan be carried out by testing the antisera on inhibitor. If antibodyproduction is detected, a fusion is warranted. Lymphocytes can beobtained by removing the spleen of lymph nodes of sensitized animals ina sterile fashion and carrying out fusion.

A number of cell lines suitable for fusion have been developed and thechoice of any particular line for hybridization protocols is directed byany one of a number of criteria such as speed, uniformity of growthcharacteristics, deficiency of its metabolism for a component of thegrowth medium, and potential for good fusion frequency. Intraspecieshybrids, particularly between like strains, work better thaninterspecies fusions. Several cell lines are available, includingmutants selected for the loss of ability to secrete myelomaimmunoglobulin.

Cell fusion can be induced either by virus, such as Epstein-Barr orSendai virus, or polyethylene glycol. Polyethylene glycol (PEG) is themost efficacious agent for the fusion of mammalian somatic cells. PEGitself may be toxic for cells and various concentrations should betested for effects on viability before attempting fusion. The molecularweight range of PEG may be varied from 1000 to 6000. It gives bestresults when diluted to from about 20% to about 70% (w/w) in saline orserum-free medium. Exposure to PEG at 37° C. for about 30 seconds ispreferred in the present case, utilizing murine cells. Extremes oftemperature (i.e., about 45° C.) are avoided, and preincubation of eachcomponent of the fusion system at 37° C. prior to fusion can be useful.The ratio between lymphocytes and malignant cells is optimized to avoidcell fusion among spleen cells and a range of from about 1:1 to about1:10 is commonly used.

The successfully fused cells can be separated from the myeloma line byany technique known by the art. The most common and preferred method isto choose a malignant line which is Hypoxthanine Guanine PhosphoribosylTransferase (HGPRT) deficient, which will not grow in anaminopterin-containing medium used to allow only growth of hybrids andwhich is generally composed of hypoxthanine 1×10⁻⁴ M, aminopterin 1×10⁻⁵M, and thymidine 3×10⁻⁵ M, commonly known as the HAT medium. The fusionmixture can be grown in the HAT-containing culture medium immediatelyafter the fusion 24 hours later. The feeding schedules usually entailmaintenance in HAT medium for two weeks and then feeding with eitherregular culture medium or hypoxthanine, thymidine-containing medium.

The growing colonies are then tested for the presence of antibodies thatrecognize inhibitors. Detection of hybridoma antibodies can be performedusing an assay where the inhibitor is bound to a solid support andallowed to react to hybridoma supernatants containing putativeantibodies. The presence of antibodies may be detected by "sandwich"techniques using a variety of indicators. Most of the common methods aresufficiently sensitive for use in the range of antibody concentrationssecreted during hybrid growth.

Cloning of hybrids can be carried out after 21-23 days of cell growth inselected medium. Cloning can be performed by cell limiting dilution influid phase or by directly selecting single cells growing in semi-solidagarose. For limiting dilution, cell suspensions are diluted serially toyield a statistical probability of having only one cell per well. Forthe agarose technique, hybrids are seeded in a semi-solid upper layer,over a lower layer containing feeder cells. The colonies from the upperlayer may be picked up and eventually transferred to wells.

Antibody-secreting hybrids can be grown in various tissue cultureflasks, yielding supernatants with variable concentrations ofantibodies. In order to obtain higher concentrations, hybrids may betransferred into animals to obtain inflammatory ascites.Antibody-containing ascites can be harvested 8-12 days afterintraperitoneal injection. The ascites contain a higher concentration ofantibodies but include both monoclonals and immunoglobulins from theinflammatory ascites. Antibody purification may then be achieved by, forexample, affinity chromatography.

The utilization of an antibody produced in the above-described manner isaccomplished by providing antibody on a solid support and passing theproducts of the non-selective lysis step over the solid support. Whereantibody is coupled to a solid support, it may be covalently orpassively bound. The solid support is typically glass or a polymer, themost commonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinyl chloride or polypropylene. The solid support maybe in the form of tubes, beads, discs or microplates. Antibody couplingprocesses are well-known in the art as described by Quash, U.S. Pat.Nos. 4,419,444 and 4,217,338, and Forrest et al., U.S. Pat. No.4,659,678, hereby incorporated by reference.

To perform the non-selective lysis protease K is used (e.g. Sigma, St.Louis, Mo., U.S.A., catalogue No. p4914 "Proteinase K"). In thisreaction, 5 μl of whole blood is added to 20 μl of PK mix (10 mM Tris pH8.0, 10 mM EDTA, 0.5% Tween 20, 0.5% NP40, and PK). A high concentrationof protease K (2.5 mg/ml) is used for 5 minutes at 55° C. to completelydegrade cellular as well as plasma protein and expose cellular nucleicacid for amplification. So that amplification may be carried out,protease K is inactivated by heating the mixture at 95° C. for 5minutes.

The reaction can thereafter be added to a small column containing theantibody on the solid support. The inhibitors will bind to the antibody.Repeated passes of the reaction mixture will eliminate the inhibitors.

Example 9

In this example, the presence of the diluent and lysis agent used forthe selective lysis step (see FIG. 1) is examined with respect toamplification inhibition. Aliquots of different amounts of ISOTON®II, adiluent manufactured by Coulter Diagnostics, and the commercialselective cell lysis agent ZAP-OGLOBIN® (also manufactured by CoulterDiagnostics) were added to standard (20 μl) PCR reactions in differenttubes using the SK38/39 system (see above). Three standard controlreactions were run in separate tubes: i) a no nucleic acid targetcontrol, ii) a no primer control, and iii) a positive control (nodiluent or lysis agent). PCR was carried out for 30 cycles. The PCRproducts were thereafter electrophoresed (agarose) and visualized withethidium bromide staining (FIG. 9, lanes 1-7). The three above-namedcontrol reactions were electrophoresed in lanes 1, 2 and 3,respectively. As expected, no product is visible in lanes 1 and 2(negative controls), while a strong product band corresponding to theexpected 115-mer is apparent in lane 3 (positive control). The lanes(lanes 3 and 4) corresponding to tubes where ISOTON®II was added at 25%and 10%, respectively, also show a strong product band corresponding tothe expected 115-mer. By contrast, the lane (lane 6) corresponding totubes where ZAP-OGLOBIN® was at 5% show no product, and the lane (lane7) corresponding to tubes where ZAP-OGLOBIN® was at 0.5% shows a veryfaint product band. It is apparent that the lysing agent containspolymerase inhibitors and that, after selective lysis, it is desiredthat less than 0.5% should be present in the tube when amplification isinitiated.

Example 10

In this example, the steps of Mode Ib (see FIG. 1) of the method of thepresent invention are performed to obtain nucleic acid from whole blood.Different amounts 4×10³, 2×10³, 2×10², 2×10¹, 2, 0.2 and 0) ofHIV-infected H9 cells were added to 0.1 ml of whole blood. For eachamount of H9 cells, the steps are carried out in the same reactionvessel (e.g. Eppendorf tube). The blood was diluted (1:10) withISOTON®II (Coulter Diagnostics) and the red cells were selectively lysedby the addition of 4 μl of ZAP-OGLOBIN® (Coulter Diagnostics). The whitecells were concentrated by centrifuging the lysate to create a whitecell pellet and a supernatant. The supernatant was removed and thepellet was resuspended in ISOTON®II (i.e. the cells were washed).Following a second centrifugation and wash, the cells were pelleted andthereafter lysed by the addition of protease K (50° C. for 1 hour). Theprotease was inactivated by heating at 95° C., for 10 minutes.

Aliquots of the lysate were added to standard (20 μl) PCR reactions indifferent tubes using the SK38/39 system (see above). Two standardcontrol reactions were run in separate tubes: i) a no nucleic acidtarget control, and ii) a positive control (10⁹ copies of 115-mer in a100 μl PCR reaction). PCR was carried out for 30 and 40 cycles. The PCRproducts were thereafter electrophoresed (agarose) and visualized withethidium bromide staining (FIG. 10A, 30 cycles; FIG. 10B, 40 cycles).The two above-named control reactions were electrophoresed in lanes 8and 9, respectively. As expected, no product is visible in lane 8(negative control), whether 30 cycles or 40 cycles were used. A strongproduct band corresponding to the expected 115-mer is apparent in lane 9(positive control). The lanes corresponding to tubes where HIV-infectedH9 cells were added in decreasing amounts (lanes 1-7) show acorresponding decrease in product band signal. Signal falls off atapproximately 2 cells (see lane 5, FIG. 10B) (the signal in lane 6 maybe due to carryover). It is clear that viral sequences can be recoveredand thereafter amplified from whole blood according to Mode Ib of themethod of the present invention.

Example 11

In this example, the steps of Mode Ia and Mode Ib (see FIG. 1) of themethod of the present invention are performed to obtain nucleic acidfrom whole blood using a filter. Different amounts (0, 1, 5 and 10 μl)of stored (21 day old) whole blood were diluted in 1 ml of ISOTON®II(Coulter Diagnostics) and the red cells were selectively lysed by theaddition of 4 μl of ZAP-OGLOBIN® (Coulter Diagnostics). The lysate wasthereafter filtered through a LOPRODYNE® filter (Pall, Glen Cove, N.Y.)nylon membrane filter in a multi-welled filtration manifold ("TheMinifold I", Schleicher & Schuell, Keene, N.H., U.S.A.) (the 10 μlsamples would not filter completely). The immobilized white cells on thefilter were thereafter washed (3×) with 250 μl ISOTON®II. The filterswere then removed and transferred to 0.5 ml Eppendorf tubes. Theimmobilized cells were lysed by the addition of protease K (2.5 mg/ml,55° C. for 10 minutes) to create a second lysate. The protease wasinactivated by heating at 95° C. for 10 minutes.

Aliquots of the protease K lysate were added to 100 μl PCR reactions indifferent tubes using the using primer pair GH26/GH27 to produce a242-mer product (see above). Two sets of PCR reactions were set up: 1)no inhibitor treatment (Mode Ib, FIG. 1); and 2) inhibitor treatment(Mode Ia, FIG. 1). Inhibitor treatment involved the addition of bovinetransferrin and the cofactor sodium bicarbonate prior to amplification.Two standard control reactions were run in separate tubes: i) a positivecontrol (human placental DNA); and ii) a no nucleic acid target control.PCR was carried out for 35 cycles. The PCR products were thereafterelectrophoresed (agarose) and visualized with ethidium bromide staining(FIG. 11A, inhibitor treatment; FIG. 11B, no inhibitor treatment). Thetwo above-named control reactions were electrophoresed in lanes marked"P" and "N", respectively. As expected, no product is visible with thenegative control. A strong product band corresponding to the expected242-mer is apparent in the positive control. The lanes corresponding to(duplicate) tubes where different amounts (1, 5 and 10 μl) of wholeblood cells were added in (lanes 3-4, 5-6 and 7-8, respectively) show acorresponding increase in product band signal, with and withoutinhibitor treatment (compare 11A with 11B). Thus, it is clear that withadequate washing on a filter, Mode Ib is an alternative to Mode Ia.

Example 12

In this example, the steps of Mode Ia (see FIG. 1) of the method of thepresent invention are performed to obtain nucleic acid from whole bloodin a low copy system (HIV). Whole blood (100 μl) was diluted in 1 ml ofISOTON®II (Coulter Diagnostics) and the red cells were selectively lysedby the addition of 4 μl of ZAP-OGLOBIN® (Coulter Diagnostics). The whitecells were concentrated by centrifuging the lysate to create a whitecell pellet and a supernatant. The supernatant was removed and thepellet was resuspended in ISOTON®II (i.e. the cells were washed).Following a second centrifugation and wash, the cells were pelleted andthereafter lysed by the addition of protease K (5 mg/ml, 55° C. for 1hour). The protease was inactivated by heating at 95°-100° C. for 10minutes.

Different amounts (2×10³, 10³, 10², 10¹, 5, 1 and 0) of HIV-infected H9cells were then added to the protease K lysate. For each amount of H9cells, the steps are carried out in the same reaction vessel (e.g.Eppendorf tube). Thereafter bovine transferrin and the cofactor sodiumbicarbonate were added to each tube, along with PCR reagents to a finalvolume of 100 μl. PCR reactions were carried out using the SK38/39system (see above). Two standard control reactions were run in separatetubes: i) a no nucleic acid target control, and ii) a positive control(10⁹ copies of 115-mer in a 100 μl PCR reaction). PCR was carried outfor 35 cycles. The products were electrophoresed and visualized byoligonucleotide hybridization analysis and autoradiography (FIG. 12).The two above-named control reactions were electrophoresed in lanes 1and 9, respectively. As expected, no product (only radiolabelled probe)is visible in lane 1 (negative control). A strong product bandcorresponding to the expected 115-mer is apparent in lane 9 (positivecontrol). The lanes corresponding to tubes where HIV-infected H9 cellswere added in increasing amounts (lanes 2-8) show a correspondingincrease in product band signal. Signal falls off at approximately 5cells (there is no signal in lanes 2 or 1, corresponding to 1 and 0cells, respectively). It is clear that viral sequences can be recoveredand thereafter amplified from whole blood according to Mode Ia of themethod of the present invention.

Example 13

In this example, the steps of Mode III (see FIG. 1) of the method of thepresent invention are performed to obtain nucleic acid from whole blood.

Whole blood samples were lysed with protease K (500 μg/ml, 55° C. for 1hour). The enzyme was inactivated by heating (95°-100° C., 10 minutes).Aliquots corresponding to 25,000, 10,000, 5,000, 2,000, 1000, 500 and 0cells were made. Bovine transferrin (250 μg) was added to each sample aswell as the cofactor sodium bicarbonate (adjusted to 10 mM) and thereaction was incubated at room temperature for 30 minutes. ConcentratedPCR reagents were then added to each sample to a final volume of 100 μl.Three standard control reactions were run in separate tubes: i) a nonucleic acid target control, ii) a no primer control, and iii) apositive control (0.2 μg of human placental DNA). In addition, threeadditional tubes (corresponding to 25,000, 5,000 and 0 cells) were leftuntreated (no transferrin or cofactor). PCR was performed for 35 cyclesusing globin primer set KM-29/HRI-12. The PCR products were thereafterelectrophoresed (agarose) and visualized with ethidium bromide staining(FIG. 13, lanes 1-13). The three above-named control reactions wereelectrophoresed in lanes 11, 12 and 13, respectively. As expected, noproduct is visible in lanes 11 and 12 (negative controls), while astrong product band corresponding to the expected 174-mer is apparent inlane 13 (positive control). The lanes (lanes 4-10) corresponding totubes where inhibitor treatment was performed show a decreasing productband corresponding to the decreasing amount of cells. By contrast, thelanes (lanes 1-3) corresponding to tubes where no inhibitor treatmentwas performed show no product. It is apparent that the inhibitortreatment is critical for amplification.

Example 14

In this example, the steps of Mode Ib and Mode III (see FIG. 1) of themethod of the present invention are compared for sensitivity andspecificity.

Mode Ib

Different amounts (10⁴, 10³, 10², 10¹, and 0) of HIV-infected H9 cellswere added to 1 ml of whole diluted blood. For each amount of H9 cells,the steps are carried out in the same reaction vessel (e.g. Eppendorftube). The blood was diluted (1:10) with ISOTON®II (Coulter Diagnostics)and the red cells were selectively lysed by the addition of 4 μl ofZAP-OGLOBIN® (Coulter Diagnostics). The white cells were concentrated bycentrifuging the lysate to create a white cell pellet and a supernatant.The supernatant was removed and the pellet was resuspended in ISOTON®II(i.e. the cells were washed). Following a second centrifugation andwash, the cells were pelleted and thereafter lysed by the addition ofprotease K (54° C. for 1 hour). The protease was inactivated by heatingat 95° C. for 10 minutes.

Mode III

Different amounts (2×10⁴, 2×10³, 10³, 5×10², 2×10², 10², 5×10, 10 and 0)of HIV-infected H9 cells were added to 50 μl of whole blood. For eachamount of H9 cells, the steps are carried out in the same reactionvessel (e.g. Eppendorf tube). Whole blood samples were lysed withprotease K (500 μg/ml, 55° C. for 1 hour). The enzyme was inactivated byheating (95°-100° C., 10 minutes). Bovine transferrin was added to eachsample as well as the cofactor sodium bicarbonate and the reaction wasincubated at room temperature for 30 minutes.

No Blood Controls

Different amounts (10⁴, 10³, 10², 10¹, and 0) of HIV-infected H9 cellswere added to uninfected H9 cells in 1 ml of ISOTON®II and were furtherprocessed for comparison.

Amplifications

Both HLA and HIV PCRs were run as described in earlier examples.Standard control reactions were run in separate tubes. The PCR productswere thereafter electrophoresed (agarose) and visualized with ethidiumbromide staining (FIG. 14A) or they were electrophoresed (PAGE) andvisualized with OH and autoradiograph (FIG. 14B). Suprisingly, theaddition of transferrin improves the specificity of the reaction (seeFIG. 14A, compare lanes 3-17 of a' with 1-16 of b'), i.e. the lanescorresponding to tubes where inhibitor treatment was performed (ModeIII) show a clean product band while the lanes corresponding to tubeswhere no inhibitor treatment was performed (Mode 1b) show nonspecificamplification of a number of products. It is apparent that the inhibitortreatment has some impact on amplification specificity.

Sensitivity is examined in FIG. 14B. The no blood controls are in lanes1-6 (see FIG. 14B, a'). A signal is seen corresponding to 1 HIV-infectedcell. Mode 1B results are shown in FIG. 14B (b') and Mode III resultsare shown in FIG. 14B (c'); both show that a signal can be detected withas little as one HIV-infected cell.

The ability of transferrin to overcome inhibition extends even toamplification involving thermal cycling. It is not clear how transferrinis able to continue functioning after exposure to such hightemperatures.

Example 15

In this example, different sources of transferrin are examined. FIG. 15is a (direct print) photograph of an ethidium bromide stained gel ofelectrophoresed PCR-amplified, HIV sequences following inhibitortreatment with rat (lanes 2-4), rabbit (lanes 5-7) and bovine (lanes8-10) transferrin in the presence of the cofactor sodium bicarbonate. Itis clear that rat transferrin does not work as an interfering reagent.

Example 16

In this example, transferrin and serum albumin are compared in themethod of the present invention. Whole blood was prepared according toMode III (see FIG. 1).

Whole blood samples (100 μl) were lysed with protease K (5 mg/ml, 55° C.for 1 hour). The enzyme was inactivated by heating (95°-100° C., 10minutes). Four sets of reactions were then set up involving the additionof interfering reagent to the protease lysate: 1) transferrin with 10 mMsodium bicarbonate; 2) transferrin with 20 mM sodium bicarbonate; 3)serum albumin with 10 mM sodium bicarbonate; 4) serum albumin with nocofactor. These reaction were performed at room temperature for thirtyminutes using a concentration range for transferrin and serum albumin(10, 25, 50, 75, 100, 150, 200 and 250 μg). PCR was then set up usingthe primer pair GH26/GH27 to produce a 242-mer product (see above). Twostandard control reactions were run in separate tubes: i) a no nucleicacid target control; and 2) a positive control (human placental DNA).Six additional control lanes were run: 1) no inhibitor treatment (twolanes); 2) 10 mM sodium bicarbonate only (one lane); 3) 50 μgtransferrin with 10 mM sodium bicarbonate (two lanes). PCR was carriedout for 35 cycles. The PCR products were thereafter electrophoresed(agarose) and visualized with ethidium bromide staining. The twoabove-named control reactions were electrophoresed in lanes 9 and 10 ofFIG. 16A, respectively, as well as lanes 17 and 18 of FIG. 16B,respectively. In FIG. 16C, the positive control is in lane 1 and thenegative control is in lane 2. As expected, no product is visible withthe negative control. A strong product band corresponding to theexpected 242-mer is apparent in the positive control.

FIG. 16A shows the impact of transferrin as a function of concentration.Lanes 1-8 of FIG. 16A correspond to 10, 25, 50, 75, 100, 150, 200 and250 μg of transferrin in the presence of cofactor. No product is visiblewith 10 μg transferrin (lane 1), while a faint product band is visiblewith 25 μg (lane 2). Strong product bands are visible at the higherconcentrations (lanes 3-8). Interestingly, however, product bands arenot as apparent with the higher concentration of the cofactor (lanes11-18).

FIG. 16A shows the impact of serum albumin as a function ofconcentration. Lanes 1-8 of FIG. 16B correspond to 10, 25, 50, 75, 100,150, 200 and 250 μg of serum albumin without cofactor. No product isvisible in any of these lanes. Serum albumin with cofactor, however,does show product (lanes 9-16). While a weak product band is visiblewith 25 μg (lane 9), strong product bands are visible at the higherconcentrations (lanes 10-16).

FIG. 16C shows the results of the additional control lanes. Lanes 3-4 ofFIG. 16C correspond to no inhibitor treatment and no product bands areevident. No product is visible with 10 mM of cofactor alone (lane 5) orwith 50 μg of transferrin alone (lane 6). Strong product bands arevisible with 50 μg of transferrin with 10 mM cofactor (lanes 7-8).

From the above it should be clear that a cofactor is critical for bothtransferrin and serum albumin to overcome polymerase inhibitors.Interestingly, the cofactor concentration must be selected low enough sothat it is not inhibiting.

Example 17

In this example, transferrins and serum albumins are examined bySDS-PAGE (see above for SDS-PAGE protocol). After electrophoresis, theprotein bands were visualized with Coomassie blue (Sigma) in 50%methanol and 10% trichloroacetic acid for 1 hour followed by destainingin 5% methanol and 7% acetic acid (FIG. 17). Lanes 1, 2 and 14 aremolecular weight markers. M1 is bovine albumin. M2 is egg albumin. M3 istrypsinogen. Lanes 3-7 are commercial serum albumin preparations. Lanes8-13 correspond to transferrins from different sources (human, horse,bovine, mouse, rat, and rabbit, respectively).

It is clear that all of the bovine serum albumin preparations are nearlyuniform. On the other hand, the transferrins are different from theserum albumins (i.e. higher molecular weight) as well as from oneanother.

Example 18

In this example, transferrins and serum albumins are examinedimmunologically (see Ouchterlony protocol). A solution of antibodyreactive with bovine serum albumin (Sigma, St. Louis, Mo., U.S.A.) wasplaced in the center well ("0") and solutions of the relevant testsamples were placed separately in the surrounding wells. Wells 1 and 2contained commercial preparations of bovine serum albumin. Wells 3-8contained commercial preparations of transferrins from different sources(human, horse, bovine, mouse, rat, and rabbit, respectively). Theinspection of the petri dish revealed the presence of precipitin linesonly with the serum albumin wells). This indicates that the antibodyonly reacted with serum albumin and not transferrin. It is clear thatall of the transferrins are different from the serum albumins.

Example 19

In this example, the effect of biochemical treatment of hematin iscompared with biochemical treatment of the non-metal-containingporphyrin "protoporphyrin." Stock solutions of 500 μM hematin (Sigma) or500 μM protoporphyrin (Sigma) were used to set up four reactions induplicate: 1) hematin only, 2) hematin with 50 μg bovine transferrinwith cofactor, 3) hematin with 100 μg bovine transferrin with cofactor,4) protoporphyrin only, 5) proto-porphyrin with 50 μg bovine transferrinwith cofactor, and 6) protoporphyrin with 100 μg bovine transferrin withcofactor. Following the reaction (30 mintes at room temperature) PCRreagents were added to the tubes and standard (20 μl) PCR reactions werecarried out using the SK38/39 system (see above). Three standard controlreactions were run in separate tubes: i) a no nucleic acid targetcontrol, ii) a no primer control, and iii) a positive control (nohematin). PCR was carried out for 30 cycles. The PCR products werethereafter electrophoresed (agarose) and visualized with ethidiumbromide staining (FIG. 19). The three above-named control reactions wereelectrophoresed in lanes 1, 2 and 3, respectively. As expected, noproduct is visible in lanes 1 and 2 (negative controls), while a strongproduct band corresponding to the expected 115-mer is apparent in lane 3(positive control). The "hematin only" tubes (lanes 4-5) show noproduct; PCR is completely inhibited. Similarly, the "protoporphyinonly" tubes (lanes 10-11) show no product; PCR is again completelyinhibited. On the other hand, product is visible in all of the lanes(lanes 6-9) representing reactions where interfering reagent was added.The ability of transferrin to overcome inhibition of anon-metal-containing porphyrin suggests that this is not mediated byiron-binding.

Example 20

In this example, the role of cofactors in the inhibitor treatment of thepresent invention is further demonstrated. Whole blood was preparedaccording to Mode III (see FIG. 1). Whole blood samples (20 μl) werelysed with protease K (0.5 mg/ml, 55° C. for 1 hour). The enzyme wasinactivated by heating (95°-100° C., 10 minutes). Reactions were thenset up involving the addition of bovine transferrin or serum albuminwith various cofactors, including bicarbonate ion, azide ion,thiocyanate ion, cyanate ion, oxalate ion, malonate ion, glycinate ion,and thioglycolate ion. These reaction were performed at room temperaturefor thirty minutes. PCR was then set up using the using primer pairGH26/GH27 to produce a 242-mer product (see above). PCR was carried outfor 35 cycles. The PCR products were thereafter electrophoresed(agarose) and visualized with ethidium bromide staining. A summary ofthe results is provided in Table 1. The presence of product bands ("+")indicated that the cofactor was in conjunction with transferrin or serumalbumin, able to overcome inhibition.

Example 21

In this example, Mode II of the present invention was performed. Wholeblood (1, 2, and 5 μl) was spotted onto a variety of membranes usingmicropipets (see Table 2). After spotting, each filter was air driedthen sat at room temperature for 6 days prior to amplification.

The digestion and amplification experiment was run on duplicate samplesof the 1 μl spot using all filters. The filters were processed asfollows. Each filter was placed in a 0.5 ml Eppendorph tube followed byprotease K treatment. This consisted of 7.5 μl diluent, 5 μl 10× PKbuffer (100 mM Tris, pH 8.0; 10 mM EDTA; 5% Tween 20; 5% NP 40), 25 μlprotease K (5 mg/ml), and 12.5 μl water. The tubes were heated at 55° C.for 5' then 95° C. for 10' then cooled to room temperature. Followingdigestion and inactivation of the protease K, PCR and sample preparationreagents were added to each tube (10 μl 10× PCR buffer, 1.5 μl 12.5 mMdNTP stock, 1 μl 10 μM primer RS-134, 1 μl 10 μM primer RS-135, 0.5 μl10 units/μl Taq stock, 5 μl 0.2M NaHCO₃, 5 μl 50 μg bovine transferrinstock, and 26 μl water). The samples were then amplified (in thepresence of the filter) for 35 cycles (95° C. 30"; 55° C. 30"; 72° 60")then analyzed on a 3% Nusiev/1% agarose gel.

Example 22

In this example, Mode Ib and III of the present invention was performedon panels of clinical, whole blood, HIV-serotested samples. PCRreactions were carried out using the SK38/39 system (see above).Standard control reactions were run. PCR was carried out for 35 cycles.The products were electrophoresed and viualized by oligonucleotidehybridization analysis and autoradiography. Table 3 is a summary of thedata. Both Mode Ib and III results show excellent correlation with priorart techniques.

Example 23

In this example, Mode III of the present invention was performed incomparison to boiling methods.

Experiment 1

Whole blood (200 μl) was boiled for 10 minutes then briefly centrifugedto precipitate the solid coagulated mass which formed. The supernatantlayer was then used directly for PCR amplification (1, 2, 3, 4, or 5μl), either with or without transferrin. Results showed (data not shown)that the samples without transferrin were inhibited when the sample sizeexceeded 1 μl, while the samples with transferrin provided signal up toand including the 3 μl aliquot. This shows that transferrin relieves theeffect of residual inhibitors present in the boiled only samples. Analiquot of 5 μl whole blood processed by Mode III gave a very intensesignal, indicating the advantage of the method over the boiling method,which provided no signal at all with a 5 μl aliquot.

It should be stressed that the heat sensitivity of inhibitors isdetermined by the nature and structure of the particular inhibitor. Forexample, where an inhibitor is a protein, thermal denaturation occurstypically at approximately 62°-65° C. On the other hand, where aninhibitor is a protein with a prosthetic group (e.g. the prostheticgroup of hemoglobin is heme), or the inhibitor is a salt (e.g. an ironsalt), thermal treatment typically requires much higher temperatures.

Experiment 2

Duplicate samples of whole blood were placed in Eppendorf tubes theneither boiled (in buffer) or digested with Protease K according to ModeIII. The samples were then prepared for PCR (100 μl) and cycled 25, 30,35 and 40 cycles. Both samples contained transferrin during the PCR. Inall cases, the boiled samples failed to provide detectable product,while the digested (Mode III) samples provided increased signal at eachpoint from 30 cycles on (data not shown). Adequate transferrin waspresent to normalize all inhibiting compounds present in the (1 μl)sample volume (when processed by Mode III).

Example 24

In this example, deoxyribonucleic acid was made from ribonucleic acidaccording to the present invention. FIG. 20 schematically shows oneembodiment of the method of the present invention for makingdeoxyribonucleic acid (DNA) from ribonucleic acid (RNA). First, reversetranscriptase is used to make cDNA from RNA. Then amplification of theDNA is carried out.

To provide HIV-RNA for development of the sample preparation methods,the plasmid pBKBH10S, which contains the complete HIV DNA sequence (8927bp) except for the replication region, was used as a template for invitro HIV RNA synthesis using the T7 phage promoter. The synthesized (+)strand of HIV RNA was used as for the sample preparation experiments.Reverse transcription conditions were studied and optimized forobtaining cDNA from the RNA using avian myeloblastosis virus (AMV-RT)and random hexamer primers. PCR amplification of the cDNA was performedusing primer pair SK-38/SK-39 to provide the usual 115-mer amplicon.This region encompasses nucleotides 1514-1541/1628-1655 of the conservedgag region. Following PCR, the 115-mer was detected by solution oligomerhybridization using the 41-mer probe SK-19.

The step of lysing the whole blood for release of RNA for conversion tocDNA is artful. It is important to release the RNA under conditionswhere it is not degraded due to simultaneous release of nucleases. Theprotease K must degrade the nucleases faster than the nucleases degradethe RNA, or if not, there will be no "net RNA" left for transcription byRT. In this experiment, free RNA was added to whole blood, and thenlysis was carried out. To minimize the RNA loss, RNAsin is added (whichis a potent RNAse inhibitor) during the protease K step. Salt (KCl) andDTT concentration are very important. For optimum results the followingreactions conditions were found:

RNA

RNA mix: 250 mM DTT (1 μl), 40 U/ml RNAsin (1.25 μl), 5 mg/ml protease K(2.5 μl), 10× PK buffer (2.5 μl), 1.3 ng tRNA+(4.0) μl), water (3.75μl), RNA (5.0 μl)

Blood

1 μl whole blood

Digest→.increment.55° C./5' then 95°/5'

Digested Blood

RT→add 15 μl RT mix:4XRT (5 μl)

Example 25

In this example, deoxyribonucleic acid was made from ribonucleic acidwhich was recovered according to one embodiment of the samplepreparation method of the present invention. FIG. 21 schematically showsthe steps of this embodiment. Basically, the method of obtainingribonucleic acid comprises first providing a sample suspected ofcontaining ribonucleic acid. This sample is mixed with a purification"reagent" comprising guanidinium thiocyanate (GSCN) andbeta-mercaptoethanol. It is preferred that the reagent is buffered (e.g.Tris-Sodium Acetate) and contains carrier nucleic acid (e.g. tRNA). Themixture is heated, preferrably above 60° C., and more perferrably to 65°C.

At this point in the method, RNA can be directly alcohol precipitatedand recovered. No centrifugation step is employed prior toprecipitation. The preferred alcohol is isopropanol.

Following the addition of alcohol, precipitation is facilitated bycentrifugation. Thereafter, the recovered RNA is washed with alcohol andthe pellet is resuspended.

The method of FIG. 21 was used to evaluate HCV clinical samples. Plasma(100 μl) was pipetted into each non-siliconized 1.5-2.0 ml microfugetube. In this case, 400 μl of the following reagent was added to eachtube:

    ______________________________________                                        400 μl                                                                            5M GSCN/0.125M Tris-HCl pH 7.5/0.3125M NaAC                             5 μl                                                                             beta-mercaptoethanol                                                    1.5 μg                                                                           t-RNA as carrier                                                       ______________________________________                                    

This mixture was vortexed for 10 seconds and incubated at 65° C. for 10minutes (mixing briefly at 5 minutes). The mixture was then cooled to 4°C. on ice (one minute).

To recover the RNA, 1 ml isopropanol was added to the mixture at roomtemperature. This was then microfuged at top speed for 10 minutes.Following centrifugation, the supernatant was removed and discarded. Thepellet (containing the RNA) was washed gently by adding 1 ml of 70%ethanol, microfuging at top speed for 5 minutes, and again removing anddiscarding the supernatant. The pellet was then resuspended in 50 μl H₂O and mixed by vortexing. The resuspension was then centrifuged for afew seconds to remove insoluble material.

Once RNA was recovered, this was amplified (5-10 μl of the resuspension)by a coupled RT/PCR amplification reaction using a thermostable DNApolymerase having endogenous reverse transcriptase activity ("rTthRT/PCR Kit", Perkin Elmer Cetus, Norwalk, Conn.) and the productsdetected with ethidium bromide on agarose gels. The procedure for thecoupled RT/PCR assay was as follows:

Reaction Mix

    ______________________________________                                        H.sub.2 O                   6.3 μl                                         10XRT Rxn. Buffer (100 mM Tris-HCl (pH 8.3),                                                              2.0 μl                                         900 mM KCl                                                                    10 mM MnCl.sub.2 -0.85 mM final concentration                                                             1.7 μl                                         dNTP: 2 mM each dATP, dCTP, dGTP, & dTTP (in                                                              2.0 μl                                         H.sub.2 O, pH 7.0)                                                            RT "downstream" primer KY78 (1.5 μM in H.sub.2 O) -                                                    2.0 μl                                         3 picomole/rxn.                                                               PCR "upstream" primer KY80 (1.5 μM in H.sub.2 O) -                                                     2.0 μl                                         3 picomole/rxn.                                                               rTth DNA polymerase: 2.5 units/ul in                                                                      2.0 μl                                         1X Enzyme storage buffer = 20 mM Tris-HCl (pH                                 7.5), 100 mM KCl, 0.1 mM EDTA, 1 mM DTT, 0.2%                                 Tween (Pierce Surfactants), 50% glycerol (v/v)                                Template nucleic acid: less than 250 ng total in TE,                                                      2.0 μl                                         10 mM Tris 1 mM EDTA)                                                         Total Reaction volume       20.0 μl                                        ______________________________________                                    

The mixture should be kept on ice before thermal cycling.

Cycler Conditions

(For use with TC9600 DNA thermal cycler):

16 minutes at 70° C. (1 min. preheat & 15 RT rxn).

1 minute at 95° C.

15 sec at 95° C. and 20 sec at 60° C. for 2 cycles

15 sec at 90° C. and 20 sec at 60° C. for 38 cycles

4 min. at 60° C.

15° C. hold

Sterile MicroAmp tubes (Perkin-Elmer-Cetus) were used. No oil overlaywas necessary.

The method of the present invention was compared by preparing known HCVsamples in parallel with the commercially available IsoQuick protocol(Microprobe Corp., Bothel, Wash.). In addition, the method was comparedby preparing samples in parallel following the substitution of sodiumiodide (NaI) for guanidinium thiocyanate. Sodium iodide has been used toisolate DNA from human serum. See M. Ishizawa et al., Nucleic Acids Res.19:5792 (1991). The results of the three procedures are shown in Table 7below:

                  TABLE 7                                                         ______________________________________                                        RNA Preparation From Plasma                                                           Amplification Results                                                 Sample    IsoQuick  The Invention                                                                              Sodium Iodide                                ______________________________________                                        1         positive  positive     negative                                     2         positive  positive     negative                                     3         negative  negative     negative                                     4         positive  positive     negative                                     5         negative  positive     negative                                     6         negative  positive     negative                                     7         positive  positive     negative                                     8         positive  positive     negative                                     Control   negative  negative     negative                                     Control   negative  negative     negative                                     ______________________________________                                    

For ease of reference, the discrepancies between the method of thepresent invention and the commerically available IsoQuick method arehighlighted in Table 7. In this experiment, the method of the presentinvention proved to have better sensitivity.

Interestingly, the sodium iodide procedure failed completely. Clearly,these chaotropes can not be substituted for each other.

Example 26

In this example, nucleic acid was recovered according to anotherembodiment of the sample preparation method of the present invention. Inthis example, the method was tested by assaying pathogen nucleic acid inspinal fluid (CSF).

The assay involved the transfer of 500 μl of CSF into a 2.0-2.5 mlmicrofuge tube. Each sample was spiked with 20 μl of buffer (TE)containing a known quantity of Lyme spirochete (Borrelia burgdorferi).See D. H. Persing et al., Science 249:1420 (1990). The reagent (500 μl)was added containing the following:

5M GSCN/0.125M Tris-HCl pH 7.5/0.3125M NaAC

1.25% beta-mercaptoethanol

2.0 μg/ml human placental DNA

This mixture was vortexed for 10 seconds and incubated at 65° C. for 10minutes (mixing briefly at 5 minutes). For comparison, sodium iodide(NaI) was again substituted for GSCN.

To recover nucleic acid, 1 ml isopropanol was added to the mixture. Thiswas then microfuged at top speed for 10 minutes. Followingcentrifugation, the supernatant was removed and discarded. The pellet(containing the nucleic acid) was washed gently by adding 1 ml of 70%ethanol, microfuging at top speed for 5 minutes, and again removing anddiscarding the supernatant. The pellet was washed a second time byadding 1 ml of 100% ethanol, microfuging at top speed for 5 minutes, andagain removing and discarding the supernatant. The pellet was thenresuspended in 50 μl buffer (TE) and mixed by vortexing.

PCR was performed using the following primer pair:

    DDO2 5'-CCCTCACTAAACATACCT-3' (18-mer)                     (SEQ ID NO:12)

    DDO6 5'- (20-mer)

The PCR Master Mix (total volume of 80 μl) contained the following:

44.5 μl de-ionized water

20.0 μl 50% glycerol

10.0 μl 10× Taq buffer

2.5 μl 10 mM dNTP

1.0 μl 50 μM DDO2

1.0 μl 50 μM DDO6

1.0 μl Taq enzyme

The Master Mix was vortexed; 80 μl was added to each tube containing a20 μl pellet of nucleic acid. This mixture was vortexed, spun andamplified. The PCR reaction involved a 95° C. denature step (5 minutes),followed by 50 cycles (95° C.--25 second hold; 55° C.--25 second hold).At the end of cycling, an extension step (72° C., 10 minutes) wasemployed. The reaction was stored in the cold until detection.

The detection assay was performed in a microtiter plate coated with thefollowing probe:

    DDO4 5'-CCCGTAAGGGAGGAAGGTAT-3' (20-mer) (SEQ ID NO:13)

At the time of assay, 100 μl neutralization/hybridization solution and25 μl of the amplified product denatured by NaoH were added to theplates for hybridization for 1 hour (37° C.). Thereafter, the wells werewashed (5×) and 100 μl of the conjugate solution was added and incubated(15 minutes, 37° C.). The wells were again washed (5×). Substratesolution (100 μl) was added and incubated (10 minutes at RT). Finally astopping solution (100 μl) was added and the reaction was read (450 nm).

The results of the three procedures are shown in Table 8 below:

                  TABLE 8                                                         ______________________________________                                        DNA Preparation From CSF                                                                 Amplification Results (450 nm)                                     Spirochete   The Invention                                                                             Sodium Iodide                                        ______________________________________                                        5000         2.515       2.697                                                500          2.630       2.523                                                100          2.548       2.079                                                 50          1.961       1.881                                                 20          0.510       0.075                                                 0           0.069       0.065                                                ______________________________________                                    

For ease of reference, the discrepancy between the method of the presentinvention and the sodium iodide procedure are highlighted. In thisexperiment, the method of the present invention proved to have bettersensitivity; at lower amounts of spirochete DNA, the method of thepresent invention was still able to provide a positive signal.

Example 27

In this example, deoxyribonucleic acid was made from ribonucleic acidwhich was recovered according to one embodiment of the samplepreparation method of the present invention. The method of the presentinvention was compared as in Example 25 (above) by preparing known HCVplasma samples in parallel with the commercially available IsoQuickprotocol (Microprobe Corp., Bothel, Wash.). Once RNA was recovered, thiswas amplified (5-10 ul of the resuspension) by a coupled RT/PCRamplification reaction using a DNA polymerase with endogenous reversetransciptase activity ("rTth RT/PCR Kit", Perkin Elmer Cetus, Norwalk,Conn.) and the products detected with ethidium bromide on agarose gels.The procedure for the coupled RT/PCR assay was as described in Example25.

FIGS. 23A and 23B show ethidium bromide stained gels of electrophoresedPCR-amplified, HCV sequences following recovery of RNA and synthesis ofcDNA from patient plasma (run in duplicates). The results of theIsoQuick method are simply indicated above the gel (i.e., the actualIsoQuick gel results are not shown). For ease of reference, positive (+)and negative (-) indicated for the method of the invention as well. Apositive (+) was scored if both duplicates showed an amplified product(244MW amplicon). Lane markers ("M") and negative ("N") and positive("P") controls were also run.

From the results in FIG. 23, it is clear that the method of the presentinvention proved to have better sensitivity. A number of known HCVpositive samples gave negative results with the IsoQuick method butshowed positive results with the method of the present invention.

Example 28

In this experiment two improvements were made to the method of thepresent invention. First, the method of recovering RNA was changed inthe hope to get less variation (e.g. to get results where bothduplicates have the same result every time) and the amplificationprotocol employed the dUTP/UNG sterilization protocol(Perkin-Elmer-Cetus).

To improve the recovery method, precipitation with isopropanol was donewith less isopropanol (500 μl or a 1:1 ratio) and at a lower temperature(4° C.) (compare with FIG. 21). It was theorized that this would resultin a better RNA yield.

Once RNA was recovered, this was amplified (5-10 μl of the resuspension)by the coupled RT/PCR amplification procedure described in Example 25with the following modification: 200 μM dATP, dCTP and dGTP were usedwith either 100 μM, 200 μM or 400 μM UTP.

FIGS. 24A and 24B show ethidium bromide stained gels of electrophoresedPCR-amplified, HCV sequences following recovery of RNA and synthesis ofcDNA [run with a low copy ("L") and high copy ("H") spike]. Theleft-hand side of the gel involves no preparation; the RNA was spikedinto buffer and directly amplified. The right-hand side of the gel showsthe results where RNA was spiked into human plasma, recovered by themethod of the present invention, and amplified with increasingconcentrations of dUTP. FIG. 24A shows a 5 μl load on the gel while FIG.24B shows a 10 μl load on the gel.

From the results of FIG. 24, it is clear that this embodiment of themethod of the present invention has excellent sensitivity; a comparisonwith directly amplified RNA shows approximately the same signal (i.e. nosignificant signal is lost by the preparation method). Furthermore, inFIG. 24B, only one duplicate is not completely positive using the methodand this was a low copy sample using dUTP at 100 μM. Clearly, the higher(400 μM) dUTP concentration shows the best sensitivity.

From the above it is evident that the present invention provides amethod for preparing nucleic acid samples without the accompanyingdificiencies of prior art methods. Thus, present invention provides aless cumbersome nucleic acid preparation method having bettersensitivity.

All patent publications cited in this specification are hereinincorporated by reference as if each individual publication werespecifically and individually indicated to be incorporated by reference.

                  TABLE 1                                                         ______________________________________                                        The Effect of Cofactors on Serum Albumin and Transferrin                                                    Bovine                                          Cofactors             BSA     Transferrin                                     ______________________________________                                        None                  -       -                                               Sodium Bicarbonate (NaHCO.sub.3)                                                                    +       +                                               Sodium Azide (NaN.sub.3)                                                                            +       -                                               Sodium Thiocyanate (NaSCN)                                                                          +       +                                               Sodium Cyanate (NaOCN)                                                                              +       +                                               Sodium Oxalate (NaO.sub.2 CCO.sub.2 Na)                                                             +       -                                               Sodium Malonate (NaO.sub.2 CCH.sub.2 CO.sub.2 Na)                                                   +       -                                               Sodium Glycinate (NaO.sub.2 CH.sub.2 NH.sub.2)                                                      +       +                                               Sodium Thioglycolate (NaO.sub.2 CCH.sub.2 SH)                                                       +       +                                               ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        SUMMARY OF MODE II FILTER DATA                                                Membrane      Size (μ)                                                                            Spotting*     Results                                  ______________________________________                                        Pall Loprodyne                                                                              .45      ws            Good                                     Pall Loprodyne                                                                              1.2      ws            Good                                     Amersham nylon                                                                              .45      ss            Good                                     Amersham nitrocellulose                                                                     .45      c             Good                                     Whatman #1 filter paper                                                                              ws            Good                                     S&S PTFE (teflon)                                                                           .45      ws (rough side)                                                                             Good                                     Pall Biodyne A                                                                              .45      ws (1,2 μl) ss (5 μl)                                                                 Good                                     (Nylon 66)                                                                    Millipore     .2       c             Good                                     BioRad nitrocellulose                                                                       .45      ss (5 μl = c)                                                                            Fair                                     S&S PTFE (teflon)                                                                           .2       ws (rough side)                                                                             Fair                                     S&S nitrocellulose                                                                          .45      ss/c          Poor                                     S&S nitrocellulose                                                                          .2       ss/c          Poor                                     BioRad Zeta Probe                    Poor                                     Pall Biodyne B                                                                              .45      ws (1,2 μl) ss (5 μl)                                                                 V.                                       (Nylon 66)                           Poor                                     ______________________________________                                         *ws = well soaked; ss = slightly soaked; c = caked on to                 

                                      TABLE 3                                     __________________________________________________________________________    SUMMARY OF HIV CLINICAL PANEL DATA                                                                    PCR        False                                                Seropositive                                                                         Sample      HIV (+)                                                                             Data                                       Panel                                                                             Specimens                                                                           Specimens                                                                            Prep. (min.)                                                                         Method                                                                             Specimens                                                                           (+)                                                                              (-)                                     __________________________________________________________________________    1   15    2      85     I    2     0  0                                       2   12    4      85     I    4     0  0                                                        100    II   4     0  0                                       3   10    4      85     I    4     0  0                                                        100    II   4     0  0                                       4   13    5      85     I    4     0   1*                                                      100    II   4     0  1                                       5   10    3      20     II   3     0  0                                       6   10    3      10     II   3     0  0                                       7   10    4      20     I    4     0  0                                                        35     II   4     0  0                                       8   8     4      15     I    4     0  0                                                        10     II   4     0  0                                       __________________________________________________________________________     *The false negative result was confirmed both by the PCR using template       DNa prepared from PBMCs isolated by the conventional Ficoll density           gradient centrifugation, and by the virus culture assay.                 

                  TABLE 4                                                         ______________________________________                                        OPTIMIZED MODE Ia                                                             TIME                                                                          ______________________________________                                                        50 μl whole blood in 1 ml ISOTON ®II                                   ↓                                                                      RBC instantly lysed by lysing agent                                           ↓                                                      5 min           WBC pelleted by 1 min. microfuging                                            ↓                                                                      Resuspend cells in 62.5 μl PK mix*                                         ↓                                                      5 min           Release DNA template Δ 55° C.                                    ↓                                                      5 min           Inactivate PK Δ 95-100° C.                                       ↓                                                      total 15 min    Amplification**                                               before                                                                        amplification                                                                 ______________________________________                                         *PK mix (10 mM Tris pH 8.0, 1 mM EDTA, 0.5% Tween 20, 0.5% NP40, 2.5 ug/m     PK)                                                                           **Amplification reaction mixture contains transferrin and NaHCO.sub.3 ;       the final concentration for HCO.sub.3.sup.- in a PCR reaction is 10 mm.       The amount of transferrin used in a PCR reaction is 50 μg/l μl bloo     (starting) in the PK digest.                                             

                  TABLE 5                                                         ______________________________________                                        OPTIMIZED MODE Ib                                                             TIME                                                                          ______________________________________                                                        50 μl whole blood in 1 ml ISOTON  II                                       ↓                                                                      RBC instantly lysed by lysing agent                                           ↓                                                                      WBC pelleted by 1 min microfuging                                             ↓                                                      5 min           WBC washed 2X with 1 ml ISOTON ®II                                        Cells recovered by 1 min                                                      microfuging                                                                   ↓                                                                      Resuspend cells in 62.5 μl PK mix*                                         ↓                                                      5 min           Release DNA template Δ 55° C.                                    ↓                                                      5 min           Inactivate PK Δ 95-100° C.                                       ↓                                                      total 15 min    Amplification                                                 before                                                                        amplification                                                                 ______________________________________                                         *PK mix (10 mM Tris pH 8.0, 1 mM EDTA, 0.5% Tween 20, 0.5% NP40, 2.5 mg/m     PK)                                                                      

                  TABLE 6                                                         ______________________________________                                        OPTIMIZED MODE III                                                            TIME                                                                          ______________________________________                                        1 min          5 μl whole blood 20 μl PK mix*                                          ↓                                                       5 min          Release DNA template Δ 55° C.                                    ↓                                                       5 min          Inactivate PK Δ 95-100° C.                                       ↓                                                       total 11 min   Amplification**                                                before amplification                                                          ______________________________________                                         *PK mix: Made up by mixing appropriate volumes of 10x PK buffer (100 mM       Tris pH 8.0, 10 mM EDTA, 05% Tween 20, 05% NP40) and stock PK (5 mg/ml),      so that the final concentration of PK buffer is 1x and the final              concentration of PK is 2.5 mg/ml                                              **Transferrin and NaHCO.sub.3 are incorporated into the standard              amplification reaction mixture. The final concentration for                   HCO.sub.3.sup.- in a PCR reaction is 10 mM. The amount of transferrin use     in a PCR reaction is 50 μg/l μg blood in the PK digest.            

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 13                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 305 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCCAGCCCCCTGATGGGGGCGACACTCCACCATGAATCACTCCCCTGTGAGGAACTACTG60                TCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGAC120               CCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAG180               GACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCC240               GCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAGG300               GTGCT305                                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GTGCTGCAGGTGTAAACTTGTACCAG26                                                  (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CACGGATCCGGTAGCAGCGGTAGAGTTG28                                                (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 242 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GTGCTGCAGGTGTAAACTTGTACCAGTTTTACGGTCCCTCTGGCCAGTACACCCATGAAT60                TTGATGGAGATGAGGAGTTCTACGTGGACCTGGACAGGAAGGAGACTGCCTGGCGGTGGC120               CTGAGTTCAGCAAATTTGGAGGTTTTGACCCGCAGGGTGCACTGAGAAACATGGCTGTGG180               CAAAACACAACTTGAACATCATGATTAAACGCTACAACTCTACCGCTGCTACCGGATCCG240               TG242                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       ATAATCCACCTATCCCAGTAGGAGAAAT28                                                (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTTGGTCCTTGTCTTATGTCCAGAATGC28                                                (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 115 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       ATAATCCACCTATCCCAGTAGGAGAAATTTATAAAAGATGGATAATCCTGGGATTAAATA60                AAATAGTAAGAATGTATAGCCCTACCAGCATTCTGGACATAAGACAAGGACCAAA115                    (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CTCGCAAGCACCCTATCAGGCAGT24                                                    (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCAGAAAGCGTCTAGCCATGGCGT24                                                    (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGTTGGCCAATCTACTCCCAGG22                                                      (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGCAGTAACGGCAGACTACT20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CCCTCACTAAACATACCT18                                                          (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CCCGTAAGGGAGGAAGGTAT20                                                        __________________________________________________________________________

We claim:
 1. A method for preparing nucleic acid samples comprising, in sequential order, the steps of:a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate, and beta-mercaptoethanol, iii) an alcohol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature above 60° C.; d) adding said alcohol to precipitate said ribonucleic acid; and e) recovering said precipitated ribonucleic acid.
 2. The method of claim 1 wherein said alcohol is isopropanol.
 3. The method of claim 1 further comprising the step of cooling said reaction mixture after step (c) prior to step (d).
 4. The method of claim 3 wherein said cooling is performed until the reaction mixture reaches room temperature.
 5. The method of claim 3 wherein said cooling is performed until the reaction mixture reaches approximately 4° C.
 6. The method of claim 1 wherein said ribonucleic acid is from a pathogen.
 7. The method of claim 1 further comprising the step of amplifying said ribonucleic acid recovered in step (e).
 8. A method for preparing nucleic acid samples from human pathogens comprising, in sequential order, the steps of:a) providing in any order, i) a sample of human plasma suspected of containing the ribonucleic acid of a human pathogen, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution, to make a reaction mixture; c) heating said reaction mixture to a temperature of 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
 9. The method of claim 8 further comprising the step of amplifying said ribonucleic acid recovered in step (f).
 10. The method of claim 9 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid.
 11. A method for preparing nucleic acid samples comprising, in sequential order, the steps of:a) providing in any order, i) a plasma sample suspected of containing ribonucleic acid, ii) an aqueous buffered solution consisting essentially of guanidinium thiocyanate and beta-mercaptoethanol, iii) isopropanol, and iv) a reaction vessel; b) adding to said reaction vessel, in any order, said sample and said aqueous buffered solution; c) heating said reaction mixture to a temperature of approximately 65° C.; d) cooling said reaction mixture to a temperature of approximately 4° C.; e) adding said isopropanol to said reaction mixture in the approximate proportion of one part reaction mixture to at least one part isopropanol to precipitate said ribonucleic acid; and f) recovering said precipitated ribonucleic acid.
 12. The method of claim 11 wherein said target ribonucleic acid is from a pathogen.
 13. The method of claim 11 further comprising the step of amplifying said ribonucleic acid recovered in step (f).
 14. The method of claim 7 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid.
 15. The method of claim 7 further comprising the step of detecting said amplified ribonucleic acid.
 16. The method of claim 8 wherein said human pathogen is a viral pathogen.
 17. The method of claim 16 wherein said viral pathogen is Hepatitis C Virus.
 18. The method of claim 12 wherein said pathogen is a viral pathogen.
 19. The method of claim 13 wherein said amplifying of said ribonucleic acid is performed by adding a thermostable DNA polymerase having endogenous reverse transcriptase activity to said recovered ribonucleic acid. 